The relationship between the multimeric size of factor VIII-von Willebrand factor (FVIII-vWF) and the support of platelet adhesion to subendothelium was studied in an annular perfusion chamber, employing human renal and umbilical arteries. Commercial factor VIII concentrates containing multimers of low molecular weight that had been shown not to correct the bleeding time upon infusion into patients with von Willebrand's disease did not support platelet adhesion in the perfusion chamber. Cryoprecipitate and two experimental FVIII-vWF concentrates containing multimers of high molecular weight supported platelet adhesion. Factor VIII-vWF purified from cryoprecipitate was subdivided into three fractions of different molecular weights (6.0–14.0, 4.0–9.0, and 3.0–7.5 X 10(6) dalton). These fractions appeared to bind equally well and to be equally effective in supporting platelet adhesion. Factor VIII-vWF with multimers of low molecular weight (0.5–1.5 X 10(6) dalton) were prepared by partial reduction. Binding of FVIII-vWF to subendothelium was not impaired, and the support of platelet adhesion appeared to be more resistant to the effect of reduction than the ristocetin cofactor activity. At high shear rate (2,500 sec-1), increased platelet adhesion was observed with partially reduced FVIII- vWF. These data indicate that the ability of FVIII-vWF preparations to correct the bleeding time is reflected in enhanced platelet adhesion to subendothelium in a perfusion chamber. These data also emphasize that multimeric size is not the only factor determining whether FVIII-vWF will support platelet adhesion.

We have studied the binding of von Willebrand factor to extracellular matrices of endothelial cells and to the vessel wall of human umbilical arteries in relation to its function in supporting platelet adhesion. CLB-RAg 201, an MAb against von Willebrand factor, completely inhibits the binding of von Willebrand factor to collagen type I and type III. CLB-RAg 201 does not inhibit the binding of 'SI-von Willebrand factor to extracellular matrices of endothelial cells, to smooth muscle cells, or to the subendothelium. CLB-RAg 201 partly inhibits platelet adhesion to these surfaces, but this directly affects the interaction between von Willebrand factor and platelets and is not due to inhibition of binding of von Willebrand factor to these surfaces.Another MAb, CLB-RAg 38, does not inhibit the binding of von Willebrand factor to collagen. CLB-RAg 38 completely inhibits the binding of von Willebrand factor to extracellular matrices. CLB-RAg 38 inhibits platelet adhesion to cellular matrices completely insofar as it is dependent on plasma von Willebrand factor. CLB-RAg 38 does not inhibit the total binding of von Willebrand factor to subendothelium, as there are too many different binding sites, but it completely inhibits the functional binding sites for von Willebrand factor that support platelet adhesion.The epitopes for CLB-RAg 38 and 201 on the von Willebrand factor molecule are located on different fragments of the molecule. These results indicate that von Willebrand factor binds to subendothelium and matrices of cultured cells by a mechanism that is different from that by which it binds to collagen.

The role of divalent cations in platelet adherence to deendothelialized human arteries in flowing blood was investigated in an annular perfusion chamber. Spreading of platelets on the subendothelium was impaired below 30 microM of free Ca2+ ions (Ca2+). When Ca2+ was replaced by Mg2+, adherence was unchanged in perfusates without exogenous factor VIII-von Willebrand factor (FVIII-vWF), but the ability of FVIII-vWF to support platelet adherence was lost. Binding of FVIII-vWF to the vessel wall was independent of divalent cations, but bound FVIII-vWF was only able to mediate adherence after exposure to Ca2+. Pretreatment of FVIII-vWF with the calcium chelator EGTA (10 mM) resulted in loss of the ability to facilitate platelet adherence, while the ristocetin cofactor activity remained intact. Full restoration of the ability to mediate platelet adherence could only be obtained by prolonged dialysis against Ca2+ in the millimolar range. These data indicate that divalent cations have at least two separate roles to play in supporting platelet adherence: (1) platelet spreading on the subendothelium requires Ca2+ or Mg2+; (2) FVIII-vWF should be exposed to Ca2+ to obtain its optimal biologic activity in supporting platelet adherence.

To study platelet activation as a phenomenon that may precede development of angiopathy in diabetes mellitus, we compared platelet adhesion and thrombus formation in a flow system with blood from insulin-dependent (type I) diabetic subjects with and without macroangiopathy and age- and sex-matched control subjects. Adhesion and thrombus formation on matrix of cultured human endothelial cells (ECM) and adhesion on matrix of human fibroblasts (FBM) were studied after exposure to flowing blood at shear rates of 300 and 1300 s-1 and exposure times of 1, 3, 5, and 10 min (and 20 min in adhesion experiments). Blood was anticoagulated with trisodium citrate (1:10 vol/vol, 110 mM) or low-molecular-weight heparin ([LMWH] 20 U/ml). Endothelial cell cultures were either unstimulated or stimulated with 4 beta-phorbol 12-myristate 13-acetate (PMA) 16 h before isolating their matrix. Platelet adhesion on ECM and FBM in citrated and LMWH-anticoagulated blood was identical in diabetic patients and control subjects, with comparable increases of adhesion with increasing perfusion times. Platelet aggregate formation on ECM of PMA-stimulated cells with LMWH-anticoagulated blood was similar in diabetic patients, whether macroangiopathy was present, compared with control subjects. Fibrin deposition and fibrinopeptide A generation during perfusion were comparable in diabetic and control subjects. Platelet thromboxane B2 formation after stimulation with arachidonic acid was increased in diabetic patients without macroangiopathy compared with age- and sex-matched control subjects. In the perfusion system, the patterns of platelet adhesion and aggregate formation on extracellular matrix in flowing blood of diabetic patients (with or without macroangiopathy), and healthy age- and sex-matched control subjects followed a similar pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

Activated blood platelets interact with fibronectin through it to the glycoprotein IIb-IIIa(GPIIb-IIIa)-complex. The cell attachment site of fibronectin with its crucial arg-gly-asp-(-ser) (RGD(S))sequence is involved in this binding. We have studied the importance of this interaction for the fibronectin dependence of platelet adhesion under flow conditions. An RGDS-containing hexapeptide (GRGDSP) was compared with a non-reactive control peptide (GRGESP). The GRGDSP-peptide inhibited thrombin induced aggregation and adhesion under static conditions at 0.1 mM. This concentration had no effect on platelet adhesion to nonfibrillar collagen type I in flow. GRGDSP at 1 mM had a significant inhibitory effect at 1500 s™1 (8.8 ± 1.4 111In platelets* 105 /cm2, versus 19.8 ± 0.5 for the control). At lower shear rates of 800 and 300 s™1 , where platelet adhesion is also fibronectin dependent, no significant differences were obtained (respectively 11.7 ± 1.1 versus 15.2 ± 2.1, and 11.4 ± 1.0 versus 13.1 ± 0.7).The relation between GPIIb-IIIa and fibronectin dependence was investigated with platelets of a patient with Glanzmann’s thrombasthenia and monoclonal antibodies to GPIIb-IIIa, using endothelial cell matrix (ECM) as a surface. Platelets of normal controls or a patient with Glanzmann’s thrombasthenia showed a inhibition of adhesion in fibronectin free plasma, after the ECM had been preincubated with anti-fibronectin F(ab’)2, of respectively _J5 and 30 percent at 300 s™1 , and 43 and 65 percent at 1300 s™1 . Incubation of platelets with anti GPIIb-IIIa showed inhibition of platelet adhesion at high shear rates. Dependence on fibronectin for platelet adhesion was still observed, even though separate experiments had shown that these anti GPIIb-IIIa antibodies could block binding of radiolabeled fibronectin to thrombin activated platelets. These data suggest the existence of a second binding system from the RGD/GPIIb-IIIa system separate for the interaction of platelets with fibronectin, which may only function when fibronectin is present on a surface.