ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats-13) cleaves von Willebrand factor (vWF) at the Tyr1605-Met1606 bond of the central A2 domain. Inability to cleave vWF results in thrombotic thrombocytopenic purpura (TTP), which is characterized by profound thrombocytopenia and microangiopathic hemolytic anemia with various degrees of organ involvement. Previous studies have demonstrated that coagulation factor VIII (

Cao et al,
PNAS
,
105
:
7416
–21,
2008
) or platelets (
Shim et al,
Blood
,
111
:
651
–7,
2008
) can accelerate proteolytic cleavage of multimeric vWF in solution by ADAMTS13 under mechanic induced shear stresses. However, the concentrations of factor VIII or platelets required to achieve a detectable increase in the proteolytic cleavage product (350K) were beyond the physiological ranges. Therefore, the physiological significance of these findings remained to be determined. In this study, we assessed whether factor VIII and platelets, both of which bind vWF with high affinity, cooperatively affected the proteolytic cleavage of vWF by ADAMTS13 under high shear stress. All experiments were performed with a fixed concentration of vWF (150 nM) and ADAMTS13 (25 nM), and various concentrations of factor VIII and platelets in a buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl and 2 mM CaCl2. The reaction mixtures (total volume 20 μl in a 0.2 ml PCR tube) were subjected to vortexing at 2,500 rpm in MixMate (manufactured by Eppendorf), which generates laminar shear stress of approximately 30 dynes/cm2. We showed that in the absence of factor VIII, lyophilized platelets at the concentration of 1,000×109 per liter did not increase the proteolytic cleavage of plasma-derived vWF by ADAMTS13. An addition of factor VIII (1, 2, and 5 nM) to the reaction mixture markedly accelerated the rate enhancing effect of lyophilized platelets in a concentration-dependent manner. In the presence of physiological concentration of factor VIII (1–2 nM), the proteolytic cleavage product (350K) detected by Western blot under non-reducing conditions reached the maximal intensity at the platelet concentration of 100×109 per liter. B-domain deleted factor VIII (FVIII-SQ) showed the similar cooperativity with platelets accelerating proteolytic cleavage by ADAMTS13. However, a construct with the acidic region (a3) that binds vWF with high affinity removed (FVIII-2RKR) did not show any cooperativity with platelets in vWF proteolysis by ADAMTS13. We conclude that binding of factor VIII and platelets to vWF cooperatively accelerates the proteolytic cleavage of vWF by ADAMTS13 under high (arterial) shear stress. These findings provide novel insight into the regulation of ADAMTS13-mediated vWF proteolysis by a coagulation factor and platelets under physiological conditions. The data also suggest a potential role of factor VIII and platelets in the therapeutic regimen for TTP.

Topics:
adamts13 gene, blood platelets, cytokinesis, factor viii, proteolysis, shear stress, von willebrand factor, mechlorethamine, blood coagulation factors, buffers

Disclosures: No relevant conflicts of interest to declare.

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2008, The American Society of Hematology
2008
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