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Shear stress activation of platelets and von Willebrand's high molecular weight multimers
Suppl. VI, 1986
THROMBOSIS RESEARCH
139
275 INHIBITION OF VON WILLEBMND FACTOR BINDING TO ACTIVATED PLATELETS BY THE TETRAPEPTIDE ARG-GLY-ASP-SER AND THE DODECAPEPTIDE OF FIBRINOGEN GAMM4 CHAINS. HR Gralnick, SB Williams, and J Hawiger. National Institutes of Health, Bethesda MD and New England Deaconess Hospital and Harvard Medical School, Boston, MA. When human platelets are stimulated by purified human thrombin, the von Willebrand factor (vWf) binds to the glycoprotein IIb/IIIa (GPIIb/IIIa). It has been shown that both fibrinogen and fibronectin can inhibit the thrombin-induced binding of vWf, We have tested the tetrapeptide analogue of the cell attachment site of fibronectin (RGDS) and the dodecapeptide of the gamma chain of fibrinogen (amino acids 400-411) ability to inhibit thrombin-induced vWf binding to platelets. Both the RGDS and dodecapeptide are competitive inhibitors of vWf binding, and both peptides totally inhibited binding. RGDS inhibited vWf binding with an IC50 of 7 PM, while the IC50 of the dodecapeptide was 110 pM. Our data suggest that the RGDS peptide has a higher affinity for the vWf binding site on thrombinstimulated platelets than does the dodecapeptide. These findings are in good agreement with the recent data from cloning of the human vWf which localize the RGDS site in vWf while no comparable dodecapeptide or analogous site has been identified (Sadler, et 31. Proc. Natl. Acad. Sci. 82:6394, 1985). The RGDS site on vWf appears to be the major attachment 7 site to thrombin-activated platelets.
276 SHEAR STRESS ACTIVATION OF PLATELETS AND VON WILLEBRAND'S HIGH MOLECULAR WEIGHT MULTIMERS. J.R. O'Brien, G.P. Salmon. Central Laboratory, St. Mary's Hospital, Portsmouth, Hants. PO3 6AC, England. Platelet adhesion to subintima is abnormal in von Willebrand's disease (VWD) only in high shear situations. Our device permits detailed study of shear forces in the absence of subintima. Air at a controlled pressure forces (e.g. heparinised) blood through tortuous fine channels in a glass fibre filter. The rate of filter blocking, e.g. cumulative drop count of the issuing blood, and the % of platelets and white cells retained in the filter can be monitored. At 5 mm Hg with normal blood the filter is not blocked in 60+ sets and 62 + 17% of platelets are retained. At 40 mm Hg blocking occurs PRP at 40 mm Hg in 26 5 8 sets and 79 + 10% of platelets are retained. initially runs through-fast but blockage occurs in 24 -+ 6 sets. EDTA blood does not block. Conclusion, supported by EM: only at high shear rate are platelets activated and aggregate (and ? stick to glass). This phenomenon is Ca dependent, is independent of red blood cells and subintima; perhaps white Blood from 16 patients with VWD did not block. Adding cells contribute. cryoprecipitate rich in R:Ag and high M.Wt. multimers corrected. Concentrates rich in FVIII and R:Ag but with no high M.Wt. multimers had no effect. Giving Conclusion: high M.Wt. multimers of VW DDAVP to VW patients also corrects. factor are required for the aggregation of shear activated platelets. This kind of shear-induced stickiness of platelets may be of physiological importance.
THROMBOSIS RESEARCH
139
275 INHIBITION OF VON WILLEBMND FACTOR BINDING TO ACTIVATED PLATELETS BY THE TETRAPEPTIDE ARG-GLY-ASP-SER AND THE DODECAPEPTIDE OF FIBRINOGEN GAMM4 CHAINS. HR Gralnick, SB Williams, and J Hawiger. National Institutes of Health, Bethesda MD and New England Deaconess Hospital and Harvard Medical School, Boston, MA. When human platelets are stimulated by purified human thrombin, the von Willebrand factor (vWf) binds to the glycoprotein IIb/IIIa (GPIIb/IIIa). It has been shown that both fibrinogen and fibronectin can inhibit the thrombin-induced binding of vWf, We have tested the tetrapeptide analogue of the cell attachment site of fibronectin (RGDS) and the dodecapeptide of the gamma chain of fibrinogen (amino acids 400-411) ability to inhibit thrombin-induced vWf binding to platelets. Both the RGDS and dodecapeptide are competitive inhibitors of vWf binding, and both peptides totally inhibited binding. RGDS inhibited vWf binding with an IC50 of 7 PM, while the IC50 of the dodecapeptide was 110 pM. Our data suggest that the RGDS peptide has a higher affinity for the vWf binding site on thrombinstimulated platelets than does the dodecapeptide. These findings are in good agreement with the recent data from cloning of the human vWf which localize the RGDS site in vWf while no comparable dodecapeptide or analogous site has been identified (Sadler, et 31. Proc. Natl. Acad. Sci. 82:6394, 1985). The RGDS site on vWf appears to be the major attachment 7 site to thrombin-activated platelets.
276 SHEAR STRESS ACTIVATION OF PLATELETS AND VON WILLEBRAND'S HIGH MOLECULAR WEIGHT MULTIMERS. J.R. O'Brien, G.P. Salmon. Central Laboratory, St. Mary's Hospital, Portsmouth, Hants. PO3 6AC, England. Platelet adhesion to subintima is abnormal in von Willebrand's disease (VWD) only in high shear situations. Our device permits detailed study of shear forces in the absence of subintima. Air at a controlled pressure forces (e.g. heparinised) blood through tortuous fine channels in a glass fibre filter. The rate of filter blocking, e.g. cumulative drop count of the issuing blood, and the % of platelets and white cells retained in the filter can be monitored. At 5 mm Hg with normal blood the filter is not blocked in 60+ sets and 62 + 17% of platelets are retained. At 40 mm Hg blocking occurs PRP at 40 mm Hg in 26 5 8 sets and 79 + 10% of platelets are retained. initially runs through-fast but blockage occurs in 24 -+ 6 sets. EDTA blood does not block. Conclusion, supported by EM: only at high shear rate are platelets activated and aggregate (and ? stick to glass). This phenomenon is Ca dependent, is independent of red blood cells and subintima; perhaps white Blood from 16 patients with VWD did not block. Adding cells contribute. cryoprecipitate rich in R:Ag and high M.Wt. multimers corrected. Concentrates rich in FVIII and R:Ag but with no high M.Wt. multimers had no effect. Giving Conclusion: high M.Wt. multimers of VW DDAVP to VW patients also corrects. factor are required for the aggregation of shear activated platelets. This kind of shear-induced stickiness of platelets may be of physiological importance.