Discussion of Papers Presented by Drs. Colwell, Waitzman, Ross, and Hockaday

Moderator: Murray M. Bern

Dr. Kefalides: Have you tested the effects of this mitogen on the in vitro growth of skin

fibroblasts?

Dr. Ross: Yes, and it is just as effective on skin fibroblasts as it is on smooth muscle cells. It

also affects glial cells. It affects every cell that

we’ve tried it on, with the striking exception of

endothelial cells and neoplastically transformed

cells.

Dr. Kefhlides: Do you see the increase in

collagen synthesis by smooth muscle cells and by

endothelial cells during cell proliferation or

during the lag phase? Dr. Ross: The platelet factor does not

specifically increase collagen synthesis, but

increases protein synthesis. The collagen synthe- sis increase that one sees is concomitant with the

increase in general protein synthesis. Studies on

collagen synthesis with endothelial cells are in

progress. Dr. Bern: It has been commented that PGI,

is a very potent inhibitor of platelet function. It is

also a smooth muscle dilator in the small vessels.

Has it been determined whether any of the

proliferative thrusts that you discover with the

traumatized vessels are any way modulated by

PGI,, and whether the platelet phenomenon

itself can be inhibited by PGJ,‘? Dr. Ross; One needs to think in terms of at

least three points of inhibition of lesion forma-

tion. One of these is obviously inhibition of

platelet function, of which PGIz would be a very

potent inhibitor. Maintenance of endothelial

integrity would be related to PGI,, limiting

accessibility of the mitogen to the underlying

tissue. Following purification (at present, --80,000-fold), amino-acid sequencing of this

large polypeptide will allow the possibility of developing specific inhibitors of the mitogen. In

collaboration with Dr. James White, we have

studied a patient with the gray-platelet syndrome. Drs. Weiss and Goodman have stud-

ied a patient with platelet storage-pool disease. In each case, the platelets are somewhat differ-

ent in appearance but they have the common feature that by morphometric examination of

Merabolism, Vol. 28, No. 4, Suppl. 1 (April), 1979

electron micrographs, the alpha granules are

missing. Lysosomes are stored in a population of

vesicles, not in the alpha granules or the dense

bodies. The dense bodies contain calcium, ADP, ATP, and serotonin. Platelet Factor IV, the

platelet-derived growth factor, and beta throm-

boglobulin appear to be stored in the alpha

granules. Since the alpha granules are missing in

these patients, neither has beta thromboglobulin,

Platelet Factor IV, or growth activity. This data

strongly supports compartmentalization, and

makes the use of an RIA for Platelet Factor IV.

and beta thromboglobulin (and if possible for the

mitogen, once purification is finished), a poten- tially useful assay for platelet-release activation

in patients. However, the unusual characteristics

of the mitogen, Platelet Factor IV, and beta thromboglobulin make them so absorptive to so

many surfaces that assay development may be

very difhcult.

Dr. P. Ward: Does the mitogen induce

monoclonal proliferation of smooth muscle cells

or fibroblasts? Dr. Ross: I don’t accept the monoclonal

hypothesis as the only explanation of atherogen- esis. I think the data can equally well be inter-

preted in another way. If a lesion forms by

injury, proliferation, regression, repeatedly over a period of many years, then one is going to have

a population of cells that will have been selected

by the proliferative response, and this population

will probably be genetically identical. If they are

genetically identical then one would measure a

single isozyme in the lesions. But instead of

coming from one cell, that single isozyme would

be derived from a population of genetically iden- tical cells; in other words, the lesions would be

polyclonal, not monoclonal. There is a very important distinction there, because if they are

monoclonal, the implication, without any evi-

dence, is that the cells are tranformed and there- fore benign neoplasms. If they are polyclonal, the implication is that the cells are not trans-

formed, and the lesions are a hyperplastic response. Obviously, approach toward therapy and understanding lesion generation is going to

423

424 DISCUSSION

be strikingly different, depending upon which of those is correct. We suggest that most lesions are

hyperplastic and not neoplastic. I cannot see a

potential role for a mitogen in a transformed cell.

We are examining growth of smooth muscle cells from patients who have had vessels removed for

surgery for varying reasons, and comparing cell

growth with that of cells from adjacent, nonin-

volved areas and lesion areas. It is difficult to get

the smooth muscle cells to grow out of the

lesions, suggesting that they have undergone a form of senescence, hardly characteristic of the

way a neoplastic cell would respond in culture.

Cells grow out beautifully from the immediately adjacent tissue, which has no lesion area. This

data is not compatible with the notion of a

transformed cell from a monoclonal lesion. Dr. Myers: Are there in vitro or in vivo

studies that have shown increased desquamation

of endothelial cells or endothelial-cell abnormal-

ities in diabetics that may lead to increased

platelet adhesion? Dr. Colwell: In cell cultures, some endothe-

lial cells have aldose reductase activity and accu-

mulate sorbitol from glucose. Thus a metabolic

lesion certainly can be postulated in the diabetic

state. Dr. Bern: Dr. Hockaday, in your studies of

platelet counts, fibrinogen, and prothrombin

activities over a 3-yr interval, were these patients

controlled by insulin?

Dr. Hockaduy: The majority were maturity

onset-type diabetics controlled on sulfonylureas

with only a minority on insulin. Those on sul-

fonylureas do have significantly high fibrinogen,

but I don’t think one can separate this from a

history of greater hyperglycemia. There is a report that sulfonylurea treatment in maturity

onset-type diabetics is associated with a faster rise of fibrinogen, but there is another report

contradicting that statement. Dr. Joist: I would like to expand on Dr.

Colwell’s excellent review of altered platelet function in diabetes mellitus. Virtually all of the

platelet functional abnormalities described in diabetic patients are based on in vitro measure- ments. All of these measurements show a rather substantial variability in normal individuals, most likely due to the high susceptibility of platelets to surface contact-induced functional alterations. Thus, there has been considerable

debate as to whether the results of all of these in

vitro measurements truly reflect the functional

state of the circulating platelets. The only

method currently available for measuring

platelet function in vivo is the bleeding time, but

this test has almost exclusively been used for

detecting decreased platelet function. We have

recently observed a significant shortening of the

template bleeding time in diabetic patients with

and without proliferative retinopathy (Joist J H

et al: DHEW/PUBL. No. (NIH) 78-1087: 662,

1978). This finding, thus, seems to provide

important information that supports the notion

that function of circulating platelets is altered in

patients with diabetes mellitus.

I would like to share with you some data

recently obtained in our laboratory concerning

the interaction of insulin with human platelets.

(Hajek A et al: J Clin Invest: in press, 1979)

This data may be of interest with respect to the

possibility that increased platelet function in

diabetics may result from alterations in certain

plasma factors. We have shown that human

platelets (like other formed elements of the

circulating blood) have specific receptors for

insulin. Washed human platelets, virtually free

of other contaminating blood cells, were incu- bated at 2O’C with biologically active ‘2sI insulin

in the presence and absence of unlabeled porcine

insulin for 3 hr in HEPES buffer. Specific bind-

ing of iodinated insulin by platelets occurred at

physiologic insulin concentrations, increased

progressively with time, was proportionate to the

number of platelets in the incubation mixture,

and had a pH optimum of 8. Scatchard analysis

of the binding data and dissociation studies

revealed evidence for negative cooperativity of the platelet insulin receptor. The highly specific

nature of the platelet insulin receptor was further illustrated by the finding that unlabeled

porcine insulin, and to a lesser extent catfish insulin and porcine proinsulin, were able to in-

hibit binding of iodinated insulin but not gluca-

gon, prolactin, growth hormone, or thrombin. The concentration of insulin receptors per platelet membrane unit surface area was esti-

mated at 26, which is very similar to that found with other insulin-binding cells such as erythro- cytes, lymphocytes, and adipocytes. No signifi- cant change in the concentration of insulin receptors was observed in platelets that had

DISCUSSION 425

undergone aggregation and the release reaction. The latter finding indicates that the platelet

storage granule membranes that are presumably

fused with the plasma membrane during the

release reaction probably do not contain insulin

receptors. The biologic role of the human

platelet insulin receptor is presently unknown.

Attempts in our laboratory to show a direct

effect of insulin even in high concentrations on

platelet function and platelet glucose metabo-

lism have so far been unsuccessful.

Dr. Rubenstein: Any ideas about the pro-

cesses that may damage endothelial cells lining

blood vessels, or that may accelerate or provoke

an injury to vessel walls and which may then be

the initial lesion in a diabetic?

Dr. Waitzman: There certainly is a latent abnormality for which diabetes is going to be the

end result and perhaps the latter can be trig-

gered by a stress phenomenon. Corticosteroids

have a tendency to block phospholipase activity

and, therefore, if you can assume its antiinflam-

matory effect during stress or otherwise, the net

result is a reduction in the release of the

substrate for prostaglandin products. Under

stress situations, released catecholamine will be

a natural blocker of adenylatecyclase activity,

and, therefore, the chronically lowered availabil-

ity of cyclic-AMP could lead to greater platelet

aggregation. Dr. Ross: Many hormones and mediators

are present in the plasma as normal constituents,

their concentration and state perhaps being

abnormal in diabetes. It is possible now to study

endothelial “injury” in cell culture. We have

considered, but not yet instituted, studies in

diabetic patients to see if there is something in

plasma, or something associated with platelets,

that may be abnormal. Obviously, if one finds

something in culture, one has to go back and find some means of assessing in vivo whether this is

simply an interesting artifact of the culture system, or whether this has some relevancy to

what is going on in vivo.

Dr. C’olwrll; Red cell microaggregates, par- ticularly in a viscous plasma system, might be

physically damaging to the endothelium. Also, there are F, receptors on the endothelium, so that one can postulate interaction of immune complexes with endothelium.

Dr. M. Peterson. Have you studied chro-

mium release from endothelial cells in culture in the presence of elevated sugar in the incubation medium?

Dr. Ross: Not yet. However. such studies are reproducible and reasonably easy.

Dr. Gabbay: How soon does the endothelium

grow back to cover a denuded area and is the

rate of reendothelialization altered by lipid

levels?

Dr. Ross: In hyperlipoproteinemic monkeys, the endothelium is altered and remains so as long

as the animals are hyperlipoproteinemic. Endo- thelial cell turnover in normal vessels is not

quiescent. There are hot spots within the aorta

and in the arterial tree of markedly elevated

levels of endothelial cell turnover that one must

assume has to do with loss and regeneration.

These hot spots appear to be increased in hyper-

lipoproteinemic experimental animals. In man,

we can only speculate about how much regenera-

tion occurs. One can hypothesize that endothe-

lial cells in vivo have a limited life span, or to put

it a better way, a limited number of cell

doublings. If the endothelium is constantly stim-

ulated to spread and double and regenerate to

cover the repair as a protective response, then

eventually the cells in that local area will run out

of cell doublings available to them, and with

increasing age, the endothelium may be incapa-

ble of effectively covering the area and unable to

regenerate. The behavior of endothelial cells in a

medium containing hyperlipemic serum. with

respect to viscosity of the cells’ plasma

membranes, is similar to that reported for other

cells. Cholesterol exchange occurs very rapidly

between the lipoproteins and the plasma

membranes. A new equilibrium establishes in a

hyperlipoproteinemic plasma with the plasma membrane, and increased numbers of cholesterol

molecules become inserted in the plasma

membrane. This leads to an increased viscosity

of the membranes of the cells, making the

membranes, in a sense, more rigid. This means

the cells are less capable of migrating and less capable of cell-shape alteration; less capable,

probably, of maintaining cell-cell attachments

and cell-connective tissue attachments. Conse- quently, they may become more liable to the normal shearing stress of the tlow of blood that goes by them.

Dr. Gabbay; Is platelet survival shortened in

426 DISCUSSION

a patient receiving a graft? Is it possible from that to estimate the process of reendothelializa- tion?

Dr. Ross: Patients before coronary bypass surgery and after bypass surgery with angio- graphic evidence of disease reportedly have markedly shortened platelet survival before surgery and normalized survival after surgery. That always amazes me to think that one small segment of a coronary artery is going to make that difference, and I’m still worried about those studies, but the data are in the literature.

Dr. Krupin: Diabetics and experimental animals with diabetes show an increased accu- mulation of intravenously administered fluores- cein. This functional abnormality occurs prior to any structural change, and may lend itself to investigation of a number of parameters that have been discussed. This early functional abnormality is reversed following treatment of the animal with insulin, or following pancreatic islet transplantation.

Dr. Forsham: In any diabetic, whether insulin-treated or not, there are huge changes in tonicity occurring in the plasma and tissue fluids. What might these simple effects on tonic- ity do to either the endothelial cells, or smooth muscle cells?

Dr. Bern: Physiologic reverberations do occur subsequent to minor changes in tonicity; such as a leukocytotic reaction, for example, and one could anticipate analogous changes in the platelet systems.

Dr. Joist: The vascular-permeability factor liberated from the platelets during the release reaction has been partially purified and charac-

terized (Nachman RL et al: J Clin Invest 51:549, 1972). However, it is not clear in which of the several different platelet storage orga- nelles this factor is located.

I would like to make a comment concerning the relationship between endothelial injury, reendothelialization, and platelet survival. It is apparent from studies with injured rabbit aorta that injury sites lose their reactivity with regard to platelet interaction within a few hours (Groves et al: Blood 50:241, 1977). This is clearly too early to be related to reendothelial- ization. The nature of the factors that cause inactivation of the site of acute vascular injury are not fully understood.

Dr. Ross: Single-injury rabbit studies are not valid in relation to whether platelet survival is going to be useful in man because the rabbit is clearly a very different model system than man. The rabbit forms no fibrin on a single injury after the balloon. In the pig and lower primates, fibrin readily forms after a single balloon injury. After the multiple-injury situation, the rabbit is very much more like the primate and the pig where one does find fibrin. 1 think platelet survival for the moment is the best index we have of endothelial injury. I’m not saying it is a good index, but it is the best one we have for the moment.

Dr. Bern: We also must be aware that within seconds after damage, a protein matrix is laid down to which the platelets thereafter attach. The phenomenon may not be surface-platelet interaction, but platelet-protein interaction, which in turn reacts to the surface.