platelet factor 4 stimulates thrombomodulin protein c-activating

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 41, Issue of October 14, pp. 25549-25556, 1994 Printed in U.S.A.

Platelet Factor 4 Stimulates Thrombomodulin Protein C-activating Cofactor Activity A STRUCTURE-FUNCTION ANALYSIS”

(Received for publication, April 21, 1994, and in revised form, June 14, 1994)

Arne SlungaardS and Nigel S . Key From the Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455

Thrombomodulin (TM) is an anionic (PI = 4) protein cofactor that promotes thrombin (THR) cleavage of protein C to generate activated protein C (APC), a potent anticoagulant. We find that the cationic platelet a-pan- ule protein platelet factor 4 (PF4) stimulates 4-25-fold the cofactor activity of rabbit TM and two differentially glycanated versions of an extracellular domain human TM polypeptide in which the glycosaminoglycan (GAG) is either present (GAG+ T M ) or absent (GAG- TM) with an ED, of 3.3-10 pg/ml. No such stimulation occurs in response to @-thromboglobulin or thrombospondin, or when protein C lacking its y-carboxyglutamic acid (Gla) domain is the substrate. Heparin and chondroitin sul- fates A and E reverse PF4 stimulation. PF4 minimally affects the Kd for THR but decreases 30-fold (from 8.3 to 0.3 p ~ ) the K,,, for protein C of APC generation by GAG+ TM. PF4 also strikingly transforms the [Ca2+] dependence profile of rabbit and GAG+ TM to resemble that of GAG- TM. A potential explanation for this is that PF4, like Ca2+, induces heparin-reversible alterations in native (but not Gla-domainless) protein C conformation as assessed by autofluorescence emission analysis. We con- clude that PF4 stimulates TM APC generation by inter- acting electrostatically with both the TM GAG and the protein C Gla domain to enhance markedly the affinity of the THRaTM complex for protein C. By this mechanism, PF4 may play a previously unsuspected role in the physiologic regulation of clotting.

Endothelial cells actively participate to maintain blood in a fluid state in part by expressing the 575-amino acid transmem- brane protein thrombomodulin (TM)’ (1,2). TM avidly (K, = 0.5 nM) binds circulating thrombin (THR) and (a) exerts a “direct anticoagulant” effect by curbing the platelet-activating and fi- brinogen-cleaving activities of THR as well as by potentiating THR inactivation by antithrombin I11 and ( b ) functions as a “cofactor” that alters the substrate specificity of THR, acceler-

94012990 and National Institutes of Health Grant R01 HL48915 (to * This work was supported by American Heart Association Grant GIA

A. S.) and by American Heart Association Minnesota Affiliate Grant 9307092 (to N. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. j: To whom correspondence should be addressed: Box 480 UMHC,

University of Minnesota, Minneapolis, MN 55455. Tel.: 612-624-9410; Fax: 612-625-6919.

The abbreviations used are: TM, thrombomodulin; THR, thrombin; APC, activated protein C; Gla domain, y-carboxyglutamic acid domain; GAG, glycosaminoglycan; PF4, platelet factor 4; GAG+ TM, human

lin modified with a GAG moiety; GAG- TM, human recombinant ex- recombinant extracellular domain (amino acids 1 4 9 7 ) thrombomodu-

tracellular domain (amino acids 1 4 9 7 ) thrombomodulin missing the GAG moiety.

ating as much as 20,000-fold its proteolytic cleavage of circulating protein C to generate activated protein C (APC) (1, 2). APC, in turn, is a powerful anticoagulant serine protease that, in conjunction with protein S, terminates the procoagulant activity of Factors Va and VIIIa. That TM-dependent activation of protein C is crucial to clotting homeostasis is shown by the fulminant, usually lethal thrombosis occurring in newborns with severe homozygous deficiency of protein C (3) and the pronounced venous and arterial thrombotic diathesis that plagues some individuals heterozygous for protein C deficiency (4).

The TM molecule is quite anionic (PI = 4, Ref. 51, in large part due to extensive posttranslational 0-linked glycanation with a chondroitin sulfate A-like glycosaminoglycan (GAG) (36). Probably because of its pronounced negative charge a t physiologic pH, a number of cationic substances such as Poly- Brene (6), poly-L-lysine (7), histidine-rich glycoprotein (81, and S-protein (8) have been found to interact strongly with TM, interfering with both its direct anticoagulant and protein C cofactor activities. To investigate a cationic proteidI“ interaction that might have pathophysiologic consequences, we recently examined the influence of three highly (PI > 11) cationic eosinophil specific granule proteins (major basic protein, eosinophil peroxidase, and eosinophil cationic protein) upon TM anticoagulant function. These cationic proteins circulate at high concentrations in the blood (9) and accumulate on endocardial surfaces (10) of patients with hypereosinophilic heart disease, which is characterized clinically by a strong propensity for thromboembolism (11). We found (12) that all three eosinophil cationic proteins are potent inhibitors of both endothelial cell- associated and soluble TM anticoagulant function. These findings delineate a potential mechanism to explain the thrombotic diathesis seen in hypereosinophilic syndromes and raise the possibility that modulation of TM anticoagulant activity by cationic proteins may play a role in the pathogenesis of human diseases.

In considering other physiologically relevant cationic proteins that might interact with TM in the vicinity of clot formation, we focused upon platelet factor 4 (PF4) as a likely candi- date. PF4 is a cationic (PI = 7.8) and hydrophobic protein that is a major constituent (20 p&/i09 platelets) of platelet a-gran- ules, which are released from platelets when they are physiologically activated by THR (13, 14). This 7.8-kDa protein has an unusually high (K, = 10-7-2 x lo-’ M (13, 14)) affinity for heparin, requiring 1.5-2 M NaCl to dislodge it from heparin- Sepharose. In addition to binding strongly to heparin, PF4 has a number of reported biologic activities including (a) antago- nizing the heparin-dependent acceleration of thrombin inactivation by antithrombin I11 (15), ( b ) inhibiting Hageman factor (factor XII) activation by negatively charged substances (16, 171, ( e ) potentiating submaximal platelet aggregation and se- cretion caused by other agonists (181, ( d ) inhibiting angiogen- esis in the chicken chorioallantoic membrane model (19), ( e )

25549

25550 Platelet Factor 4 and Thrombomodulin

TABLE I Effect of PF4 upon kinetics of TM interaction with thrombin and protein C

Kinetic parameters were calculated from data describing the thrombin dependence and protein C dependence of APC generation (see “Exper- imental Procedures”) using nonlinear regression analysis (32). Data are expressed 2 standard error.

Preparation Kd for thrombin V,, for thrombin K,,, (apparent) for protein C V,, for protein C

nM ng APC I min v ng APC I min GAG+ TM 1.20 f 0.08 5 + _ 1 8.3 t 0.8 97 f 5 GAG+ TM +33 pg/ml PF4 0.83 f 0.08 173 t 1 0.27 f 0.02 241 -c 4

serving as a “chemokine” chemotactic factor for human PMNs for human neutrophils and monocytes (20), and ( f , reversing experimental immunosuppression (21). Given the strongly inhibitory influence of other cationic proteins upon TM function (see above), we predicted that PF4 would similarly inhibit TM anticoagulant function and thus promote thrombosis in the presence of activated platelets.

EXPERIMENTAL PROCEDURES

Materials APC chromogenic substrate S-2366 and thrombin inhibitor 1-2581

were obtained from Kabi Vitrum (Franklin, OH). Bovine protein C and activated protein C were from Enzyme Research Laboratories, Inc. (South Bend, IN). Bovine plasma thrombin (2,500 NIH unitdmg protein), chondroitin sulfate A, lysozyme, cytochrome c, and protamine sulfate were obtained from Sigma. Squid cartilage chondroitin sulfate E, superspecial grade, was obtained from Seikagaku America, Inc. (Rockville, MD). Rabbit lung thrombomodulin and y-carboxyglutamic acid (Gla) (domainless protein C), generated by chymotrypsin cleavage of bovine protein C (22), were generously provided by N. L. and C. T. Esmon (Oklahoma Medical Research Foundation, Oklahoma City, OK). Hank’s buffered salt solution was obtained from Life Technologies, Inc. Defibrinated plasma was prepared by either heating or Ancrod treat- ment. Human PF4 was isolated from units of outdated human platelet packs as described previously (23) utilizing heparin-agarose affinity chromatography, except that the final stage purification was achieved by Sephadex G-100 chromotography as described previously by Deuel et al. (24) rather than by anion-exchange chromatography. The resulting PF4 ran as a single band of apparent M, 9000 on overloaded SDS- polyacrylamide gels. For key preliminary experiments (not shown) performed prior to the availability of this PF4 preparation, we used recombinant human PF4 (191, kindly provided by Ted Maione, Repligen Corp., Cambridge, MA. However, authentic human PF4 isolated from human platelets behaved identically to recombinant PF4 and was used for all the experiments reported herein. The P-thromboglobulin preparation (25) and thrombospondin (26) were isolated from supernatant of tbrom- bin-stimulated, freshly isolated human platelets, as described previously. These preparations were homogeneous as judged by SDS-polyacrylamide gel electrophoresis. Differentially glycanated versions of a recombinantly produced polypeptide fragment of human TM encompassing the entire extracellular domain (amino acids 1-497, starting at the first amino acid after cleavage of the 18-amino acid NH,-terminal leader sequence) expressed in Chinese hamster ovary cells were kindly provided by Tish Young, Berlex Biosciences (Richmond, CAI. These soluble extracellular domain polypeptides contain a Met3” + Leu mutation that confers resistance to inactivation by oxidants (271, but this mutation causes no significant alterations in such critical parameters as the Kd for thrombin, the K, for protein C, and the calcium dependence profile (27-29). Chinese hamster ovary cells transfected with pRd CMV vector containing this TM sequence produce two forms of the TM separable by anion-exchange chromatography. The first has an apparent molecular mass of 116 kDa and contains a chondroitinase ABC- cleavable GAG moiety; the second is an unglycanated version of the same polypeptide with an apparent mass of 95 kDa. Henceforth, these differentially glycanated versions of the extracellular domain TM will be referred to as, respectively, GAG+ TM and GAG- TM. These forms are analogous to two previously described pairs of differentially glycanated extracellular domain TM fragments, namely, the TMD1-105 and TMD1-75 characterized by Parkinson et al. (30) and the rsTMa and rsTMP characterized by Nawa et al. (31).

Methods Assay of Thrombomodulin APC Generation-APC generation by the

three TM preparations in the presence or absence of PF4 was assayed as described previously (12) in 96-well microtiter plates utilizing the

chromogenic substrate S-2366. Briefly, aliquots containing 500 n~ bovine protein C, 0.5 nM TM, 1 mM CaCI,, and various concentrations of purified human PF4 were mixed in 100 mM NaCl and 50 mM Tris, pH 7.4 (assay buffer), and incubated for 10 min at 37 “C prior to the addition of 2 n~ bovine thrombin. After a 10-min incubation at 37”, further generation of APC was quenched by the addition of 1 mM EDTA and 30 PM thrombin inhibitor 1-2581. APC generation was then quantitated by the addition of 500 p~ S-2366 and determination of the initial rate of chromogenic substrate cleavage as assayed at 405 nm on a Thermomax Microtiter V,, plate reader (Molecular Devices Corp., Menlo Park, CA). A standard curve constructed using defined amounts of purified bovine activated protein C was used to convert mOD/min to ng of APC/min. Another set ofwells was composed as described above except that buffer was substituted for the TM. Values obtained from these wells were subtracted from those obtained in the presence of TM to correct for TM-independent activation of protein C by thrombin, cleavage of S-2366 by any residual thrombin, and spontaneous hydrolysis of S-2366. These corrections were small in comparison with the TM-dependent signal, typically <0.75 ng ofAPC/min (cf. Fig. 1). Nearly identical results were obtained using human thrombin and protein C (eg. 20-fold stimulation of GAG+ TM APC generation by 100 pg/ml PF4 (not shown)), but because of the restricted availability of human protein C and the availability of a bovine Gla-domainless protein C preparation, our experiments were all conducted with bovine thrombin and protein C. For experiments utilizing cationic proteins other than PF4, these proteins were substituted for PF4 in the above protocol.

Polyanion Reversal of TM Cofactor Stimulation Caused by PF4--10 pg/ml PF4 was added to microtiter wells containing 0.5 nM GAG+ TM, 1 m~ CaCl,, and 500 nM protein C in assay buffer. The resultant mixture was incubated for 10 min at 37 “C, whereupon various concentrations of heparin, chondroitin sulfate E, or chondroitin sulfate A were added and the mixture was incubated for another 15 min. 2 nM thrombin was then added to initiate the generation ofAPC, which was assayed as described above. Wells containing all the components described above except PF4 were assayed in parallel. Results were expressed as a ratio of APC generated in the presence of PF4 to that generated in its absence.

Effect ofPF4 upon Thrombin and Protein C Dependence of GAG+ TM APC Generation-To determine the apparent Kd of thrombin for GAG+ TM, increasing concentrations of bovine thrombin were added to the APC assay system described above. APC generated by thrombin in the absence of TM under otherwise identical conditions was subtracted from each value obtained in its presence to correct for TM-independent cleavage of protein C at the various thrombin concentrations. To determine the apparent K, of protein C for the THR.TM complex, increasing amounts of benzamidine-free bovine protein C were added to wells containing the basic assay mixture as described above, and the generation of APC was initiated by the addition of 2 nM thrombin. Incubation was for 5 min rather than 10 min to keep APC generation linear with respect to time at the high rates of APC generation seen in the presence of high concentrations of protein C in the presence of PF4. Aparallel set of wells without GAG+ TM was used to correct APC generation for TM-independent cleavage of protein C at the various protein C concentrations. Data from the resulting thrombin and protein C dependence curves of APC generation were analyzed (Table I) with nonlinear regression analysis (Statistics, Version 5.2 Systat, Inc., Evanston, IL) to avoid the hazards of linear analysis related to error distribution (32).

Role of Protein C Gla Domain in PF4 Acceleration of TM APC Generation-Assay conditions were as for the standard assay, except that the final calcium concentration was 0.5 m ~ , the optimum for APC generation by GAG+ TM in the presence of 33 pg/ml of PF4 (see Fig. 4). In some preparations 500 n~ Gla domainless protein C was substituted for native protein C.

Calcium Dependence Profile ofAPC Generation by GAG+ TM, GAG- TM, and Rabbit TM-Conditions were identical to those in the standard assay except that Ca2+ concentration was varied as indicated. For the 0 Ca2+ value, 500 p~ EDTA was added to chelate any contaminating Ca”.

Platelet Factor 4 and Thrombomodulin 25551

A

C e .-

100

3 0

10

GAG+ I I It GAG-

“[I GAG+ in plasma 1 - I - . ’ . . - a . I . . ’ -.... I - - . - . - - I

0 1 1 0 100

PF41 (CLg/mI) FIG. 1. Influence of human PF4 upon thrombomodulin protein

C cofactor activity. 0.5 IMI rabbit TM, GAG+ TM, or GAG- TM was

in 50 nm Tris, 100 mM NaCl, 1 mM CaCl,, pH 7.4. PF4 at the indicated suspended in 96-well microtiter plate wells containing 500 nM protein C

final concentrations was added and the mixture incubated a t 37 “C for

proceed for 10 min. The reaction was quenched by the addition of 30 p~ 10 min. 2 nM thrombin was added, and APC generation was allowed to

1-2581 and 1 mM EDTA. APC was quantitated spectrophotometrically by assaying cleavage of the chromogenic substrate S-2366 at 405 nm on a V,, mictrotiter plate reader (Molecular Devices Corp., Menlo Park,

TM; 0, rabbit lung TM; 0, GAG+ TM in the presence of 15% defi- CA). Results are expressed as ng of APC/min. B, GAG+ TM; A, GAG-

brinated plasma. Error bars denote * standard deviation.

APC generated in wells containing a similar composition, but missing TM was subtracted from the value obtained in the presence of TM to correct for TM-independent cleavage of protein C by thrombin.

Autofluorescence Analysis of PF4/Protein C Interaction-The assay was modeled on that of Johnson et al. (33). 1.5-ml solutions in a 1 x 1-cm quartz cuvette containing 60 pg/ml of bovine protein C in 50 mM WIS, 100 mM NaCI, 250 1” EDTA, pH 7.4, were excited a t 280 nm in a Perkin-Elmer model MPF-44A fluorescence spectrophotometer. Scan- ning emission in preliminary experiments revealed an optimum at 337 nm. Therefore, for further experiments,. emission was followed at 337 nm. Emission obtained in the presence of 250 PM EDTA (i.e. 0 calcium) was arbitrarily assigned a value of 100%. In one set of experiments, Ca2+ was then added to a final concentration of 1 mM, and fluorescence emission was again assayed. In another set of experiments, 33 pg/ml PF4 was added to protein C in the presence of 0 Ca”, and emission was reassayed, subsequent to which heparin (IO units/ml) was added to ascertain the heparin-reversible component of the PF4 effect on protein C autofluorescence. Corrections were made for the diluent effect of adding these substances. The addition of heparin or PF4 did not shift the emission maximum of protein C from 337 nm. Heparin had no measurable autofluorescence at these concentrations. PF4, which lacks tryptophan residues, had a very low level of fluorescence (-1% that of the protein C solution), and this value was subtracted from the values obtained in the presence of both PF4 and protein C.

RESULTS To test our hypothesis that PF4, like other cationic proteins,

would inhibit the capacity of the THR.TM complex to generate APC, we determined the effect of increasing amounts of purified PF4 upon the ability of three different TM preparations to generate APC from protein C, (a) a full-length rabbit lung TM and ( b ) two versions of a recombinantly produced polypeptide fragment of human TM encompassing the entire extracellular domain that differ only in the presence (GAG+ TM) or absence (GAG- TM) of the GAG moiety. As shown in Fig. 1, rather than inhibiting APC generation (here depicted on a log scale) by these TM constructs as we had predicted, PF4 instead unex-

10

0 1 1 0 100

[protein] (pg/mI) FIG. 2. Cationic and platelet a-granule protein influence on

TM cofactor activity. Assay conditions were as described in the legend to Fig. 1 and under “Experimental Procedures,” except that the indicated proteins were substituted for PF4 at the designated concentrations. 0, thrombospondin; A, P-thromboglobulin; 0, eosinophil major basic protein (MBP); B, lysozyme; A, protamine sulfate.

pectedly stimulates AF’C generation 4-fold by rabbit TM (0) and GAG- TM (A) and 25-fold by GAG+ TM (.) at 100 pg/ml PF4. GAG+ TM was also accelerated 10-fold by PF4 in the presence of 15% defibrinated plasma (0). Pertinent controls (not shown) demonstrated that PF4 has a negligible effect upon the TM-independent activation of protein C by THR and does not affect the activity ofAPC toward the chromogenic detection substrate. The 50% effective dose (ED,,) for the PF4 acceleratory effect is 3-10 pg/ml, approximately the concentration of PF4 present in serum (13,141. Comparison of the dose-response curves of GAG+ (B) and GAG- TM (A) to PF4 reveals that presence on TM of the GAG moiety potently enhances the acceleratory effect of PF4 on AFT generation. To examine the specificity of this PF4-mediated TM cofactor

acceleratory effect, we compared its activity to that of the two other major platelet a-granule proteins that bind heparin, the P-thromboglobulin-related proteins and thrombospondin (13, 14), as well as that of other representative cationic proteins. As shown in Fig. 2, heparin-binding but anionic (PI = 4.7, Ref. 34) thrombospondin (0) has no effect on GAG+ TMAPC generation at concentrations 5 100 pg/ml. The P-thromboglobulin preparation f a group of proteolytically related polypeptides comprised of -70% low affinity PF4 (PI 8.01, 20% platelet basic protein (PI 10.5), and 10% “p-thromboglobulin antigen” (PI 7.0, Ref. 25) had a minor (2-fold) acceleratory effect discernible only at the highest concentrations tested (A). This minimal influence may be attributable to the presence in our p-thromboglobulin preparation of trace amounts of immunodetectable PF4. The eosinophilic cationic granule protein, major basic protein (0) was, as expected, strongly inhibitory. However, equally cationic lysozyme (B, PI 11) and cytochrome c (PI 10.6, not shown) failed to influence AFT generation, as did bovine serum albumin and hemoglobin (not shown). In contrast, highly cationic (>60% arginine) protamine sulfate (A) had a biphasic acceleratory effect that was maximal a t 3.3 pg/ml and decreased a t higher concentrations. Thus, PF4 is unique amongst the platelet a-granule heparin-binding proteins with regard to its striking acceleration ofAPC generation by GAG+ TM, but other cationic proteins can have inhibitory, negligible, or acceleratory effects.

25552 Platelet Factor 4 and Thrombomodulin

A 15 - e heparin

"t chondroitin sulfate E chondroitin sulfate A

10 -

5 -

€l I

0 1 1 0 100

[GAG1 (PLgW FIG. 3. Polyanionic GAG reversal of PF4 influence on TM co-

factor activity. Solutions containing 0.5 rm GAG+ TM were incubated in the presence of 10 pg/ml PF4 under conditions described in the legend to Fig. 1. After 10 min of incubation, either heparin (Dl, chondroitin sulfate E (m), or chondroitin sulfate A (A) was added, and the mixture was incubated 15 min further at 37 "C. 2 nM thrombin was then added, and APC generated over 10 min was assayed as described. An- other set of wells comprised as above but missing PF4 was also assayed for APC in parallel. Data are expressed as a ratio of APC generated in the presence of PF4 to that generated in its absence (i.e. -fold stimulation of APC generation).

Given the high afflnity of PF4 for heparin and other GAGs, it is plausible to envision PF4 binding to GAG+ TM at its chondroitin sulfate-like (36) GAG moiety. This type of electrostatic interaction should be reversed by subsequent addition of GAGs with a higher or similar affinity for PF4 than TM GAG. Fig. 3 demonstrates that the acceleration of GAG+ TM cofactor activity caused by 10 pg/ml of PF4 is potently reversed by three such GAGs, heparin, chondroitin sulfate E, and chondroitin sulfate A. In the absence of added GAG, 10 pg/ml of PF4 produces the expected 15-fold acceleration of APC generation over that seen in the absence of PF4. Subsequent addition of other GAGs reverses this acceleration with an ED,, of 0.33-1 pg/ml. As might be expected given the known relative affinities of PF4 for various GAGS (351, heparin (0) is more effective at reversing the PF4 effect than are chondroitin sulfate E (H) and chondroitin sulfate A (A).

To define the mechanism underlying PF4 stimulation of TM APC generation, we determined whether PF4 affects the apparent Kd for THR or, alternatively, the K, for protein C of APC generation by the THRvTM complex (Table I). The apparent Kd for thrombin is minimally affected by the presence of PF4, decreasing from 1.2 to 0.83 nM, but the V,, is accelerated 34-fold. In striking contrast, presence of PF4 decreases the apparent K,,, of protein C for the THR.TM complex more than 30-fold from 8.3 to 0.27 but increases the V,, only 2.5-fold. Thus, PF4 stimulation of TM protein C cofactor activity is attributable to a pronounced increase in the affinity of the THR.TM complex for its substrate, protein C, rather than altered affinity of TM for THR.

Protein C is a vitamin K-dependent coagulation factor containing Gla residues in a 41-amino-acid NH,-terminal domain of its light chain (22). Presence of this Gla domain is essential for the physiologic function of protein C because intact endothelial surfaces activate protein C lacking its Gla domain (Le. Gla-domainless protein C) at 4 % of the rate of native protein C (22), and the resulting Gla-domainless APC is an ineffective anticoagulant (22). We therefore asked whether the greatly increased affinity of the THR.TM complex for protein C, as demonstrated in the kinetic analysis above, requires the pres-

TABLE I1 Role of protein C Gla domain in PF4 acceleration of GAG+ TM

cofactor activity Conditions were as described for the standard assay (see legend to

Fig. 1) except that the final calcium concentration was 0.5 mM, the optimum for APC generation by GAG+ TM in the presence of 33 pg/ml PF4 (see Fig. 4). In some preparations, 500 nM Gla domainless protein C was substituted for native protein C. Data are expressed 2 S.D.

Preparation AFT generated

Protein C Gla domainless Drotein C

GAG+ TM nglmin

2.8 f 0.75 4.8 f 1.2

GAG+ TM +33 pg/ml PF4 57 -c 3.8 2.1 2 0.7

ence of this functionally important Gla domain. Accordingly, we compared APC generation by GAG+ TM in the presence or absence of 33 pg/ml PF4 using either native or Gla-domainless protein C (Table 11). In the absence of PF4, GAG+ TM generates more chromogenically detectable APC from Gla-domainless protein C than from an equimolar amount of native protein C, as described previously for rabbit TM (22). In the presence of 33 pg/ml PF4, however, GAG+ TM converted native protein C to APC at a 25-fold greater rate than it did Gla-domainless protein C. Presence of PF4 accelerates APC generation from native protein C 20-fold, but it diminishes APC generation from Gla- domainless protein C by 50%. These data show that PF4 stimulation of GAG+ TM APC generation requires the presence of the protein C Gla domain.

We hypothesized that PF4, known to bind avidly to heparin as well as other GAGs, might similarly bind to the GAG domain of GAG+ TM and, thereby, negate its considerable influence (7, 8,30,36,37) upon TM function. The main aspect of TM protein C cofactor activity known to be governed by GAG status is the calcium dependence profile ofAPC generation. APC generation by the THR.TM complex is strictly dependent upon the presence of Ca2+, because little or no APC is generated in its absence (30,411. GAG+ forms of TM, such as rabbit TM (30,41), TMD1- 105 (30), and our GAG+ TM show a hyperbolically increasing rate of APC generation with increasing Ca", as confirmed in Fig. 4, B and C. By contrast, GAG- forms of TM, such as the elastase-cleaved TM fragment (411, TMD1-75 (301, and our GAG- TM preparation (Fig. 4A) display an unusual, biphasic, or bell-shaped Ca2+ profile, increasing rapidly from 0 Ca2+ to an optimum at subphysiologic (0.3-0.5 a) Ca2+, decreasing there- after with higher concentrations. If the presence of PF4 functionally "deGAGs" GAG+ forms of TM, then their APC calcium dependence profiles should be transformed to resemble that of GAG- TM. As shown in Fig. 4 A , addition of 33 pg/ml of PF4 to GAG- TM accelerated APC generation at all Ca2+ concentrations, but the overall Ca2+ profile retains its characteristic biphasic nature with an optimum at subphysiologic (0.5 mM) calcium. In contrast, the Ca" dependence of GAG+ TM (panel B ) and rabbit TM (panel C) shifts markedly from the hyperbolic increasing profile seen in the absence of PF4 to a biphasic profile with an optimum at 0.5-0.75 mM Ca2+, strongly remi- niscent of the GAG- TM profile. As assessed by Ca2+ dependence, therefore, PF4 converts GAG+ forms of TM to function as GAG- forms.

An unanticipated finding from these studies (Fig. 4) was that, in the presence of PF4, both the GAG- and the GAG+ forms of TM generate considerable amounts of APC in the virtual absence of Ca2+ (0 Ca" values were obtained in the presence of 0.5 mM EDTA). This effect was most notable in the case of GAG+ TM (panel B ) , where at 0 Ca2+ almost no APC generation was detectable in the absence of PF4, but the presence of PF4 permits generation of APC at a rate more than


B

no PF4 + 33 pg/ml PF4

0 1 2 3 4 5 [calcium] (mM)

no PF4

+ 33 pglml PF4

20 - n n

U 0 0 1

0 1 2 3 4 5

[calclum] (mM)

z E . lw1f i noPF4

5 80 0 + 10 pgml PGF4

0 1 2 3 4 5 [calclum] (rnM)

generation by GAG+ TM, GAG- TM, and rabbit lung TM. Condi- FIG. 4. PF4 influence on calcium dependence profile of APC

tions were identical to those described in the legend to Fig. 1 except that the Ca2+ concentration was vaned as indicated. For the 0 Ca2+ value, 500 p~ EDTA was added. APC generated in wells containing a similar composition but missing TM was substracted from APC values obtained in the presence of TM to correct for TM-independent cleavage of protein

either 10 pg/ml (rabbit lung TM) or 33 pg/ml PF4 (GAG- and GAG+ C by thrombin. APC was measured in the absence (0) or presence (B) of

TM). Panel A, GAG- TM; Panel B , GAG+ TM; Panel C, rabbit lung TM.

6-fold greater than that achieved at any concentration of Ca2+ in the absence of PF4 (Fig. 4B).

Previous studies employing autofluorescence analysis have demonstrated that the magnitude of Ca2+-dependent changes in protein C conformation closely parallels the Ca2+ dependence ofAPC generation by the THR.TM complex (33). To explain the ability of PF4 to permit APC generation by THR.TM despite the absence of Ca2+, we investigated the possibility that PF4 might, in effect, substitute for Ca2+ by binding to the anionic Gla domain of protein C to engender similar conformational changes. To test this hypothesis, we employed autofluorescence analysis similar to that performed by Johnson et al. (33) in their investigations of calcium interactions with protein C. As shown in Table 111, when solutions containing 60 pg/ml of protein C in a Ca2+-free buffer were excited at 280 nm and emission followed at its maximum of 337 nm, the addition of 1 mM calcium, as expected, produced an approximately 10% decrement in fluorescence (33). Addition of 33 pg/ml o f PF4 instead of calcium also altered autofluorescence emission but in the opposite direction, to 105.5% of control. The ED,, for PF4 aug- mentation of protein C autofluorescence was 3-10 pg/ml (not shown), similar to its ED,, for stimulating TM cofactor activity (Fig. 1). Subsequent addition of heparin (10 unitdml), which itself had no fluorescence, immediately and completely reversed this PF4-induced increment in autofluorescence. Gla domainless protein C also exhibited a decrement in autofluorescence in response to addition of CaZ+, although it was smaller than that noted for protein C, as previously reported (33). Unlike protein C, however, Gla-domainless protein C autofluorescence showed no change upon addition of PF4 in lieu of Ca2+. Thus, PF4 causes Gla domain-dependent changes in protein C autofluorescence.

DISCUSSION

Interaction of PF4 and Other Cationic Proteins with Throm- bomoduZin-Given the strong negative charge of the TM molecule at physiologic pH, it is not surprising that a wide variety of cationic proteins have been shown to influence TM function. All cationic substances that have been investigated to date (PolyBrene, poly-L-lysine, vitronectin, major basic protein, eosinophil peroxidase, and eosinophilic cationic protein) have been found to have uniformly inhibitory effects upon TM direct anticoagulant or cofactor activity. In contrast, we now describe a massive (up to 25-fold) amplification of the cofactor activity of three different forms of TM caused by human PF4, a major platelet a-granule protein released from platelets upon physi- ological activation by thrombin. That such amplification does not occur in the presence of the two other predominant platelet a-granule proteins, thrombospondin and the p-thromboglobulin-related proteins, further highlights the unusual nature of the PF4-TM interaction. Perhaps relevant to our finding that PF4 accelerates protein C cleavage by TM-bound thrombin is the previous observation that PF4 also stimulates the proteolytic activity of another serine protease, human neutrophil elastase (38, 39).

At least three salient features of PF4 structure may, either alone or in combination, contribute to its ability to influence TM function: ( a ) cationicity, ( 6 ) the capacity to bind heparin, and (c) a-amphipathic helical structure manifested in the carboxyl terminus of the molecule (13). Cationicity per se is obvi- ously not sufficient to confer this property on molecules since other cationic proteins can strongly inhibit, fail to influence, or accelerate TM cofactor activity (Fig. 2). Similarly, heparin- binding capacity alone does not by itself confer this ability since p-thromboglobulin and thrombospondin fail to elicit this effect (Fig. 2). A combination of cationicity and an a-amphipathic helical structure may be sufficient to confer this activity be-

25554 Platelet Factor 4 and Thrombomodulin TABLE I11

Autofluorescence analysis of PF4 /protein C interaction

of 337 nm. The value obtained in the presence of no added calcium and 250 p~ EDTA was assigned a value of 100%. The indicated reagents were Solutions containing 60 pg/ml protein C or Gla domainless protein C were excited at 280 nm, and fluorescence emission followed at its maximum

then added, and emission at 337 nm was remeasured. Data are expressed as fluorescence emission as a percent that in the absence of calcium. Data are shown f standard deviation.

Preparation Fluorescence emission"

0 mM Ca2+ (EDTA) +1 m~ Ca2' +33 pg/ml PF4 +PF4 + heparin (10 unitdml)

Protein C 100 105.5 't 1.2' 99.7 't 0.6 Gla domainless protein C

89.5 t 1.4b 93.8 t O.gb 99.0 t 0.5 99.4 -c 0.7

Fluorescence emission at 337 nm as a percentage of that in 0 m~ calcium.

100

' Fluorescence magnitude significantly different from that in 0 calcium, p < 0.005.

cause, in experiments not shown, both the 14-amino acid wasp venom mastoparan and the 26-amino acid bee venom melittin amplify APC generation by GAG+ TM more than 10-fold at a concentration of 33-100 pg/ml. On the other hand, such a highly ordered structure is probably not essential for a protein to possess this activity because protamine sulfate also accelerates TM APC generation (Fig. 2). Further experiments utilizing peptide fragments of PF4 such as the 13-amino acid carboxyl terminus that mediates heparin-binding through an a-amphipathic helical structure (19) and rPF4-241 (40), a full- length mutant in which Lys to Glu sustitutions in the carboxyl terminus preserve a-amphipathic helical structure but ablate cationic heparin-binding capacity, may permit more precise def- inition of the minimal structural requirements for PF4 amplification of TM cofactor activity.

Mechanism for PF4 Stimulation of Thrombomodulin Cofac- tor Actiuity-Our studies demonstrate that PF4 interacts electrostatically with components of the THR.TM/protein C system to decrease more than 30-fold the K , of protein C for the THR.TM complex by a mechanism requiring presence of the protein C Gla domain. Thus, PF4 stimulation of TM cofactor activity is reversed by polyanionic substances such as heparin (Fig. 3) and does not occur in the presence of Gla-domainless protein C (Table 11). In addition to enhancing TM cofactor activity, PF4 also elicits a dramatic change in the calcium dependence profile of APC generation by GAG+ (but not GAG-) forms of TM, converting the hyperbolically increasing configuration characteristic of GAG+ forms of TM to resemble the biphasic subphysiologic optimum configuration characteristic of GAG- forms of TM (Fig. 4). In aggregate, these findings strongly suggest that PF4 interacts with the TM GAG domain so as to negate its influence upon TM cofactor activity, thereby render- ing the GAG+ TM functionally equivalent to GAG- TM. Indeed, the extensively characterized GAG- elastase cleavage fragment of rabbit TM (41) exhibits cofactor activity characteristics remarkably similar to those of GAG+ TM in the presence of PF4, namely: ( a ) a biphasic profile and subphysiologic calcium optimum for APC generation and ( b ) a rate of APC generation at this optimal calcium concentration 10-fold higher than that of GAG+ rabbit TM, which is attributable to a large decrease of the K, for native (but not Gla-domainless) protein C (41).

Our finding that PF4 stimulates APC generation by rabbit TM several-fold (Fig. 1) apparently contradicts that of Bourin et al. (8), who found no such effect. The probable explanation for this is that these latter experiments assayed APC generation a t a final calcium concentration of 2-2.5 mM. As shown in Fig. 4C, at these nonphysiologic calcium concentrations, our rabbit TM preparation also exhibits little or no PF4 acceleration of APC generation. Due to the unusual calcium dependence of rabbit TM in the presence of PF4, however, a t 1 mM calcium, a 4-fold acceleration is evident. Further emphasizing the potential physiologic relevance of our findings is the observation (not shown) that when the protein C concentration in our assay is decreased from 500 to 75 nM (the concentration found in human

plasma (1,211 PF4 stimulates APC generation a t 1 mM calcium 6-fold rather than 4-fold.

There are three models that have been proposed to explain the Ca2+ dependence of APC generation by TM and, therefore, three potential mechanisms whereby PF4 might influence TM Ca2+-dependence. First, Johnson et al. (33) have shown that the magnitude of protein C autofluorescence changes seen with increasing Ca2+ concentrations closely parallels the hyperbolic Ca2+ dependence ofAPC generation by (GAG+) rabbit TM. This strongly suggests that Ca2+-induced changes in protein C conformation control the rate of APC generation. If so, our finding that PF4 also causes changes in protein C conformation (Table 111) whose magnitude is affected by [Ca2+l (see below) provides one potential explanation for its capacity to influence TM cofactor Ca2+ dependence. Second, Kurosawa et al. (41) have proposed the existence of a Ca2' bridge between protein C and and TM. This proposition is supported studies by Zushi et al. (42) showing loss of bell-shaped Ca2+ dependence in a GAG- TM fragment resulting from mutation of a potential Ca2+-binding TM amino acid residue, Asp349, located at the beginning of the fourth epidermal growth factor domain known (43) to mediate protein C binding. If the TM GAG moiety extends to the vicinity of this domain, then PF4 might bind to the GAG and negate its influence in this area, thereby converting the Ca2+ dependence of GAG+ TM to resemble that of GAG- TM by "unmasking" this site. Athird suggestion (44) is that TM GAG contacts anion binding exosite 2 of thrombin to induce conformational changes that regulate its Ca2+ dependence. This hypothesis is based upon the finding by Ye et al. (45) that purified TM GAG enhances the fluorescence emission of thrombin labeled with anilinonaptha- lene-6-sulfonic acid at its active site histidine even when anion binding exosite I is occupied by binding to TM. PF4, by binding to the TM GAG moiety, could interfere with this interaction and so reverse the conformational changes in thrombin that are responsible for its hyperbolic Ca2+ dependence.

Given the close correlation between Ca2+-induced protein C conformational changes and rate of APC generation by THR.TM (see above), our autofluorescence studies (Table 111) raise the possibility that PFWprotein C interactions (in addition to the PF4/TM GAG interactions discussed above) may play a role in PF4 stimulation of TM cofactor activity. These experiments show that PF4, like CaZ+, causes a Gla domain- dependent change in protein C autofluorescence, albeit in the opposite direction. This ability of PF4 to induce changes in protein C conformation in the absence of CaZ+ may in part explain its enhancement of TM cofactor activity in the absence of Ca2+ (Fig. 4). The failure (not shown) of PF4 to accelerate APC generation by GAG+ TM in the absence of Ca2' when Gla-domainless protein C (whose autofluorescence is unaf- fected by PF4) is substituted for protein C supports this proposition. The fact that PF4 enhances, whereas Ca" decreases, protein C autofluorescence does not rule out the possibility that both engender similar conformational changes near the scissile bond that favor cleavage by THR.TM because the external


tryptophan residues responsible for protein C autofluorescence are probably not in this vicinity. In experiments not shown, we found that solutions of protein C suspended in buffers containing 0.3 and 1.0 mM calcium had a PF4-dependent increment in fluorescence of, respectively, 50 and 30% that seen in the absence of Ca". PF4 induction of protein C conformation a t physiologic Ca2+ concentrations might explain PF4 acceleration of GAG- TM cofactor activity, a setting where PF4/TM GAG interactions are precluded. The more dramatic stimulatory effect of PF4 upon GAG+ TM cofactor activity might, therefore, re- flect a synergistic interaction of the functional influences of both PF4PTM GAG and PF4/protein C binding.

Physiologic Relevance and Implications of Findings-Our findings, though based on purified TM constructs and in vitro assays, raise the possibility that PF4, a major platelet a-granule constituent released by activated platelets, may stimulate endogenous forms of TM to generate the potent natural anticoagulant APC and, thereby, play a previously unsuspected role in the physiologic regulation of clotting. Endogenous forms of TM that might interact with PF4 include the cell-associated form in vascular endothelial beds, the lower molecular weight soluble form of TM found to circulate in human blood in sur- prisingly high concentrations (up to 300 ng/ml(46,47)), and the form recently found to be associated with platelets themselves (48). Although PF4 accelerates TM cofactor activity with an ED,, of 3-10 pg/ml (Fig. 11, a range similar to that found in human serum (131, it is more plausible to envision PF4 influ- encing APC production by TM in the immediate vicinity of an activated and degranulating platelet, where local PF4 concentrations are apt to be much higher. Of note in this regard, immunofluorescent studies have shown that high concentrations of PF4 accumulate in vivo on damaged endothelial surfaces, the site of TM expression, as a result of platelet adhesion and degranulation (49). Contrary to the possibility that PF4 serves an anticoagulant function is the finding of Bourin et al. (8) that PF4 antagonizes the direct anticoagulant action of TM (i.e. its ability to prolong the thrombin clotting time). However, even if this proves to be true for the endothelial form of TM as well, the net effect of PF4 would be to permit local thrombosis on the surface of the endothelial cell but promote systemic anticoagulation by enhancing generation of APC, which would circulate freely in blood. The apparently paradoxical divergent effects of PF4 upon TM cofactor and direct anticoagulant activity might, thus, serve as a mechanism to localize thrombosis while preventing catastrophic systemic coagulation. Compati- ble with the proposal that PF4 might play a systemic anticoagulant role are previous studies demonstrating that PF4 prolongs the activated partial thromboplastin time by inhibiting activation of the contact activation system (16, 17). Further- more, in experiments undertaken to evaluate the possibility that PF4 interacts with other Gla domain-containing clotting factors in addition to protein C, we find that PF4 prolongs both tissue factor- and Russell viper venom-initiated human plasma clotting times (not shown). Further experimentation will be required to ascertain whether PF4 exerts physiologically or pharmacologically meaningful anticoagulant actions.

Regardless of their ultimate physiologic significance, however, our findings emphasize the importance of the GAG domain in TM function and implicate both the TM GAG domain and the protein C Gla domain as probable loci for interactions of other physiologically relevant cationic proteins with the TMI protein C system. Moreover, they demonstrate the utility of using cationic proteins to probe structure-function relation- ships of the TM system and invite further examination of potential interactions of PF4 (and alternative cationic proteins) with other Gla domain-containing proteins.

Acknowledgments-We thank Connie Lindor and Troy Decker for excellent technical assistance; Naomi Esmon, Oklahoma Medical Re- search Foundation, for supplying rabbit lung thrombomodulin and Gla- domainless protein C; Tish Young, John Parkinson, and Berlex Bio- sciences, Richmond, CA for supplying GAG+ and GAG- TM and insightful discussion; Ted Maione, Repligen Corp., Cambridge, MA for supplying recombinant human PF4; and Gary Nelsestuen, University of Minnesota Biochemistry Department for assistance with the autofluorescence experiments.

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