Implication of protein S thrombin-sensitive region with membrane binding via conformational changes in the gamma-carboxyglutamic acid-rich domain.

In the vitamin K-dependent protein family, only protein S (PS) contains a thrombin-sensitive region (TSR), located between the domain containing the gamma-carboxyglutamic acid and the first epidermal growth factor-like domain. To better define the role of TSR in the PS molecule, we expressed a recombinant human PS (rHPS) and its analogue lacking TSR (rTSR-less), and prepared factor Xa- and thrombin-cleaved rHPS. A peptide reproducing TSR (TSR-peptide) was also synthesized in an attempt to obtain direct evidence of the domain involvement in PS anticoagulant activity. In a coagulation assay, both rTSR-less and factor Xa-cleaved PS were devoid of activated protein C cofactor activity. The TSR-peptide did not inhibit rHPS activity, showing that TSR must be embedded in the native protein to promote interaction with activated protein C. The binding of rHPS to activated platelets and to phospholipid vesicles was not modified after factor Xa- or thrombin-mediated TSR cleavage, whereas the binding of rTSR-less was markedly reduced. This suggested a role for TSR in conferring to PS a strong affinity for phospholipid membranes. TSR-peptide did not directly bind to activated platelets or compete with rHPS for phospholipid binding. The results of the present study show that TSR may not interact directly with membranes, but probably constrains the gamma-carboxyglutamic acid-rich domain in a conformation allowing optimal interaction with phospholipids.

The platelet release reaction: granules' constituents, secretion and functions.

Although anucleated, blood platelets are highly organized cells rich in different types of organelles. Three specific granule populations store different types of constituents, some of which are at high concentrations. Platelets thus transport some specific compounds through the whole body. During circulation, platelets are reactive to various stimuli and release the materials stored in the specific granules. This 'release reaction' is an important step of primary haemostasis. Energy and messengers required for platelet reactivity are provided by mitochondria and the dense tubular system. Each granule population has specific properties concerning both the structure and the role played by the released constituents. Dense granules contain small non-protein molecules that are secreted to recruit other platelets. alpha-Granules contain large adhesive and healing proteins. Lysosomes contain hydrolases able to eliminate the circulating platelet aggregate. The extrusion of storage granules' content to the platelet's environment occurs according to regulated secretion events: movements of granules, apposition and fusion of granule and plasma membranes. Typical platelet disorders resulting from a storage granule abnormality are referred to as a storage pool defect and are characterized by a prolonged bleeding time.

A new approach in the study of the molecular and cellular events implicated in heparin-induced thrombocytopenia. Formation of leukocyte-platelet aggregates.

Heparin-induced thrombocytopenia (HIT), a relatively common complication of heparin therapy, results of platelet activation, via the receptor for the Fc domain of IgG (FcgammaRIIa), by heparin-dependent-antibodies, commonly directed against the heparin-platelet factor 4 (H-PF4) antigenic complex. Our strategy was to use whole blood allowing the study of leukocyte-platelet interactions. Experiments were performed with blood from healthy donors incubated with HIT patients' plasma and different concentrations of heparin. We showed that 75% of the HIT patients' plasma induced the formation of leukocyte-platelet-aggregates in a heparin-dependent-manner. The formation of leukocyte-platelet-aggregates induced by HIT plasma in the presence of heparin was (i) independent of the healthy blood donor FcgammaRIIa polymorphism, (ii) correlated with the levels of anti H-PF4 IgG antibodies contained in the patients' plasma, and to a lesser extent to anti H-PF4 IgM antibodies, and (iii) was mediated by P-selectin. This report opens new prospects in the study of the molecular and cellular events implicated in HIT.

Thiosulfinates inhibit platelet aggregation and microparticle shedding at a calpain-dependent step.

Thiosulfinates (TSs) are sulfur compounds generated through the processing of different Allium species with antiplatelet property. To further define this platelet inhibitory effect we studied diallyl-TS (Al2TS), dipropyl-TS (Pr2TS). and dimethyl-TS (Me2TS) on platelet responses. The three TSs inhibited dose-dependent platelet aggregation, with IC50 values of 15+/-2, 19+/-2, and 9+/-1 microM for Al2TS, Pr2TS and Me2TS, respectively. TSs had no effect on the expression of a platelet procoagulant surface, measured by flow cytometry as the binding of annexin V-FITC. They inhibited the microparticle shedding and clot retraction. Since the microparticle shedding is a calpain-activation dependent step, we assessed calpain activation by analysis of autoproteolysis in shorter active forms and by talin proteolysis in the presence of TSs. Calpain activation was inhibited by TSs independently of fibrinogen binding. Thus, TSs represent a new category of platelet inhibitors, acting on calpain-induced events.

Platelets induce human umbilical vein endothelial cell proliferation through P-selectin.

We studied whether platelets could participate in the endothelial cell monolayer regeneration in the case of a vessel damage. Incorporation of [3H]-thymidine into the DNA of human umbilical vein endothelial cells (HUVECs) was measured after 48 h of co-incubation with platelets. The effect of platelets was compared to that of platelet-free supernatants from thrombin-activated platelets that had secreted their active granule constituents. Platelets dose-dependently induced HUVEC proliferation. Platelets preactivated by thrombin induced similar proliferation as did unactivated platelets (proliferation factor = 7 - 8), indicating that preactivation of platelets was not required. Platelets fixed with paraformaldehyde had no effect, suggesting that the platelet mitogenic effect required a mobile, alive membrane. Ketanserine and suramin reduced by at most 30 % the platelet-induced proliferation; supernatants of thrombin-activated platelets caused only minor proliferation (proliferation factor = 2), suggesting that secreted 5-hydroxytryptamine and growth factors poorly contributed to the proliferative effect. When the co-incubation was performed in the presence of an anti P-selectin antibody, the platelet-induced HUVEC proliferation was inhibited. The results suggest that platelet adhesion participate in the control of the endothelial regeneration and that platelet P-selectin is a molecular determinant of the proliferative signal.

Calpain controls the balance between protein tyrosine kinase and tyrosine phosphatase activities during platelet activation.

Protein phosphorylation was studied during platelet stimulation in two ranges of ionized [Ca2+]. At ionized [Ca2+]i< or = 1 microM, proteins were phosphorylated. At ionized [Ca2+]i > or = 4 microM, phosphoproteins disappeared. Protein dephosphorylation was prevented by the combined action of calpeptin and phosphatase inhibitors. Protein tyrosine phosphatase activity was stimulated regardless of the ionized [Ca2+] level. Protein tyrosine kinase activity was stimulated at ionized [Ca2+]i < or =1 microM, whereas at ionized [Ca2+]i > or =4 microM, no protein tyrosine kinase activity was observed except in the presence of calpeptin. Thus, the massive tyrosine phosphoprotein disappearance observed at a high ionized [Ca2+]i resulted not only in protein tyrosine phosphatase activation, but also in calpain-induced protein tyrosine kinase inactivation.