Jonathan M. Gerrard

Separable assembly of platelet pseudopodal and contractile cytoskeletons

Unactivated platelets contain about 69% G actin and less than 10% of the contractile proteins in a cytoskeletal core resistant to extraction with 1% Triton X-100. Activation by thrombin leads, within 1 min, to the formation of pseudopodia and contractile gels, accompanied by the reduction of the G-actin content to about 22% and the development of cytoskeletal cores containing 70%-80% of the total actin and 60%-80% of the total myosin and actin-binding protein. Inhibition of pseudopodal formation by pretreatment with cytochalasin B before thrombin activation results in the loss of most of the actin-binding protein and about one third of the actin from the cytoskeletal core. Myosin incorporation and contractile gel formation are unaltered by this treatment. Conversely, activation with phorbol 12-myristate 13-acetate leads to pseudopodal but not contractile gel formation, with cytoskeletal cores containing mostly actin and actin-binding protein. These results demonstrate that there are separable cytoskeletal assembly processes in platelets for pseudopodal and contractile gel formation.

Platelet storage pool deficiency: diagnosis in patients with prolonged bleeding times and normal platelet aggregation

We evaluated 46 patients with prolonged bleeding times, and no demonstrable abnormalities of either von Willebrand factor or platelet aggregation, for possible deficiency of platelet storage pool. Studies of ATP release from thrombin‐stimulated platelets and enumeration of dense granules in platelet whole mounts were performed in these patients. Seventeen patients (35%) had both decreased ATP release and decreased numbers of dense granules, suggesting the presence of a storage pool defect. Thus, storage pool deficiency may be present in the absence of the classical aggregation abnormalities. Evidence of storage pool deficiency should be considered in all patients with an isolated unexplained prolongation of the bleeding time. The methods used in this study are readily applicable to most clinical laboratories.

Platelet aggregation independent of adp release or prostaglandin synthesis in patients with Hermansky-Pudlak syndrome

Platelets from patients with the Hermansky-Pudlak (HPS) syndrome are deficient in the storage pool of adenine nucleotides and serotonin. As a result, the storage pool deficient (SPD) platelets develop only single waves of clumping when stimulated by threshold concentrations of aggregating agents which cause irreversible, biphasic aggregation of normal platelets. Yet, patients with HPS either have no bleeding problems or only mild symptoms. In the present study we have evaluated the importance of prostaglandin synthesis and secretion to the irreversible aggregation of HPS platelets. Results of the study demonstrate that aspirin-treated SPD platelets, which cannot form thromboxane or undergo the release reaction on stimulation by arachidonate, can still undergo irreversible aggregation in response to thrombin and ADP if treated first with epinephrine. A mechanism of membrane modulation mediated by alpha-adrenergic receptors cooperatively linked to the endoperoxide and thromboxane receptor can secure irreversible aggregation of normal or abnormal platelets despite absence of secretion and prostaglandin synthesis.

Platelet aggregation: cellular regulation and physiologic role.

The hemostatic functions of the platelet depend in a large part on adhesion and aggregation processes. Both the proteins that mediate the adhesive processes and their cellular binding sites have been identified, with much information derived from the study of several inherited syndromes of impaired hemostasis. Implications for more effective prevention of pathologic thrombosis are stressed.

Stimulation of platelet protein phosphorylation by arachidonic acid and endoperoxide analogs

The present study has investigated the influence of arachidonate, endoperoxide analogs, and the calcium ionophore A23187 on platelet aggregation and on the phosphorylation of platelet proteins. Following stimulation of platelets by these agents a rapid increase in phosphorylation of three proteins was observed which began at the same time as the initial formation of platelet aggregates. These three proteins were the 260,000 dalton actin-binding protein, a 40,000 dalton protein in unknown function, and the 20,000 dalton myosin light chain. When extensive aggregation was reached, the extent of phosphorylation returned toward baseline. Pretreatment of platelets with aspirin completely inhibited both aggregation and protein phosphorylations induced by arachidonate, but had only partial inhibitory effects on endoperoxide analogs or A23187. Since endoperoxide analogs and A23187 may trigger endogenous production of prostaglandin endoperoxides and thromboxane A2, in addition to having a direct effect of their own, it is probable that the partial inhibition seen was due to inhibition of that component of their effect due to this endogenous production, though other effects of aspirin can not be entirely ruled out. Since recent evidence shows that phosphorylation of myosin light chain results from calcium stimulation of a protein kinase in the presence of calmodulin, the results are consistent with mobilization of calcium as the primary role of the arachidonate-endoperoxide-thromboxane pathway.

Salicylic acid inhibition of the irreversible effect of acetylsalicylic acid on prostaglandin synthetase may be due to competition for the enzyme cationic binding site

Salicylic acid (SA), a weak inhibitor of the prostaglandin endoperoxide synthetase or fatty acid cyclooxygenase enzyme, is known to prevent irreversible enzyme inhibition by acetylsalicylic acid (ASA). The interaction of arachidonic acid with ferrous sulfate was used as a model to study the reaction of the fatty acid with the postulated enzymic cationic binding site on Fe2+-heme. SA was as potent as ASA in inhibiting the cooxygenation of arachidonic acid and ferrous sulfate. The results suggests that SA could complete effectively for the enzyme cationic site with ASA. Thus SA may block ASA acetylation of the cyclooxygenase by preventing ASA from binding to this site.

Glycation of platelet protein in diabetes mellitus: lack of correlation with platelet function.

The relationship of nonenzymatic glycation of platelet proteins to altered platelet function was studied in 33 diabetic patients. Platelets isolated from diabetic patients were glycated to a greater extent than those isolated from nondiabetic controls. No relationship was found between the level of glycation of platelets in diabetics to parameters commonly used to monitor glycemic control (glycated hemoglobin, glycated albumin, fasting blood glucose). Platelets isolated from diabetics did not show an increased level of aggregation and Thromboxane B2 production as compared to nondiabetic controls. No significant relationship was found between the level of glycation and percent aggregation of platelets. The lack of a relationship between glycation and aggregation suggests that the former may not be responsible for the functional changes in platelets seen in diabetics.

Epinephrine and other activators of prostaglandin endoperoxide synthetase can reduce Fe3+-heme to Fe2+-heme.

In view of recent evidence that activation of prostaglandin endoperoxide synthetase by lipid peroxides may relate to the ability of such peroxides to reduce heme, we tested other activators of this enzyme. Epinephrine, norepinephrine, ascorbic acid and tryptophan were all found to reduce Fe3+-heme to Fe2+-heme, though tryptophan was considerably weaker than the others. We suggest that reduction of heme by these compounds might account for their ability to reduce the lag phase on addition of substrate to the enzyme. Epinephrine was assessed for its effects on the lag phase in activation of soybean lipoxygenase and was found to cause a similar reduction of the lag phase of this related enzyme. These findings support the concept that reduction of Fe3+-heme to Fe2+-heme is critical to activation of both the prostaglandin endoperoxide synthetase and soybean lipoxygenase enzymes, and that mechanisms involved in regulation of the valence of iron are important for regulating enzyme activity.

Origin and Role of Calcium in Platelet Activation-Contraction-Secretion Coupling

Agonist interaction with platelet surface receptors initiates a complex but concerted series of events inducing alterations in morphology, biochemistry, and physiology (White, 1968; Marcus, 1978; Holmsen and Weiss, 1979; Zucker and Nachmias, 1985). Intracellular move ments of calcium ions accompany and may play a critical role in regulating these biochemical and physiological processes (Statlandet al., 1969; Feinman and Detwiler, 1974; Hathaway and Adelstein, 1979; Feinstein, 1980; Gerrardet al., 1981; Foxet al., 1983; Menahsiet al., 1984). For lack of precise methods to monitor intracellular free calcium, earlier studies relied heavily on indirect methods to demonstrate agonist-induced calcium mobilization. However, synthesis of novel calcium indicators has made it possible to demonstrate agonistinduced calcium mobilization concurrent with cell activation (Rinket al., 1982; Grynkiewiczet al., 1985; Raoet al., 1985a,b).