Thrombotic thrombocytopenic purpura (TTP) is the prototypical microangiopathy characterized by disseminated microthromboses, hemolytic anemia, and ultimately organ dysfunction. A link with deficiency of the von Willebrand factorcleaving protease (ADAMTS13) has been demonstrated, but additional genetic and/or environmental triggers are thought to be required to incite acute illness. Here we report that 4 days of ADAMTS13 functional inhibition is sufficient to induce TTP in the baboon (Papio ursinus), in the absence of inciting triggers because injections with an inhibitory monoclonal antibody (mAb) consistently (n ‫؍‬ 6) induced severe thrombocytopenia (< 12 ؋ 10 9 /L), microangiopathic hemolytic anemia, and a rapid rise in serum lactate dehydrogenase. Immunohistochemical staining revealed the characteristic disseminated platelet-and von Willebrand factor-rich thrombi in kidney, heart, brain, and spleen but not lungs. Prolonged inhibition (14 days, n ‫؍‬ 1) caused myocardial ischemic damage and asplenia but not death. Control animals (n ‫؍‬ 5) receiving equal doses of a noninhibitory anti-ADAMTS13 mAb remained unaffected. Our results provide evidence for a direct link between TTP and ADAMTS13 inhibition and for a mild disease onset. Introductionvon Willebrand factor (VWF) is a multimeric glycoprotein that bridges platelets to injured arterial vessels through interactions with both subendothelial collagen and platelet membrane receptors. Unusually large VWF multimers (UL-VWFs) are released as VWF precursors into the bloodstream by stimulated endothelial cells. 1 These high-molecular-weight proteins are abnormally adhesive, being able to bind and cross-link platelets in circulation even in the absence of endothelial injury. 2 Normally, UL-VWFs are rapidly cleaved by circulating VWF-cleaving protease (AD-AMTS13), 3 which generates VWF multimers of sizes seen in normal plasma. 4 The inability to process UL-VWF in cases of ADAMTS13 deficiency can cause disseminated platelet-rich thrombi, which block terminal arterioles, 1,5 leading to hemolytic anemia with ischemic organ failure and ultimately death in patients with thrombotic thrombocytopenic purpura (TTP). Diagnosis is based on signs of concurrent thrombocytopenia with hemolytic anemia and fragmented red blood cells (schistocytes) in the absence of other identifiable primary causes. 6 ADAMTS13 deficiency can be hereditary by mutations in the ADAMTS13 gene 3 or acquired by inhibiting autoantibodies to ADAMTS13. 7 The former is currently treated by infusion of fresh frozen plasma, which contains donor ADAMTS13 to overcome the deficiency. The latter often requires plasma exchange to both replenish the diminished proteolytic activity and remove inhibitors.These plasma therapies could effectively reduce mortality to approximately 20%, 8 but morbidity still is considerable and not seldom as a consequence of the plasma therapy. 9,10 Safer therapeutic strategies are therefore required 11 and could focus on the inhibition of the platelet-VWF interaction 12 or on the reconst...

Abstract-Platelet adhesion in arterial blood flow is mainly supported by the platelet receptor glycoprotein (GP) Ib, which interacts with von Willebrand factor (vWF) that is bound to collagen at the site of vessel wall injury. Antibody 6B4 is a monoclonal antibody (MoAb) raised against purified human GPIb. MoAb 6B4 inhibits both ristocetin-and botrocetin-induced, vWF-dependent human platelet agglutination. MoAb 6B4 furthermore blocks shear-induced adhesion of human platelets to collagen I. We studied the antithrombotic effect of this inhibitory murine MoAb 6B4 in a baboon model of arterial thrombosis. When injected into baboons, intact IgG and its F(abЈ) 2 fragments caused almost immediate thrombocytopenia, whereas injection of the Fab fragments alone did not. Fab fragments were subsequently used to investigate their in vivo effect on platelet deposition onto a thrombogenic device, consisting of collagen-rich, glutaraldehyde-fixed bovine pericardium (0.6 cm 2 ), at a wall shear rate ranging from 700 to 1000 s Ϫ1 . Baboons were either pretreated with Fabs to study the effect of inhibition on platelet adhesion or treated 6 minutes after placement of the thrombogenic device to investigate the effect on interplatelet cohesion. Pretreatment of the animals with bolus doses ranging from 80 to 640 g/kg Fab fragments significantly reduced 111 In-labeled platelet deposition onto the collagen surface by Ϸ43% to 65%. Only the highest dose caused a significant prolongation (doubling) of the bleeding time. Ex vivo ristocetin-induced platelet agglutination was equally reduced. Treatment with a bolus of 110 g/kg Fab fragments after a thrombus was allowed to form for 6 minutes had no effect on further platelet deposition. We therefore conclude that Fab fragments or derivatives of inhibitory anti-GPIb antibodies may be useful compounds to prevent thrombosis.

ALX-0681 is a therapeutic

The pathophysiology of thrombotic thrombocytopenic purpura (TTP) can be explained by the absence of active ADAMTS13, leading to ultra-large von Willebrand factor (UL-VWF) multimers spontaneously interacting with platelets. Preventing the formation of UL-VWF-platelet aggregates therefore is an attractive new treatment strategy. Here, we demonstrate that simultaneous administration of the inhibitory anti-VWF monoclonal antibody GBR600 and the inhibitory anti-ADAMTS13 antibody 3H9 to baboons (prevention group) precluded TTP onset as severe thrombocytopenia and hemolytic anemia were absent in these animals. In addition, partial VWF inhibition was not enough to prevent thrombocytopenia, demonstrating the specificity of this therapeutic strategy. GBR600 treatment of baboons during acute TTP (treatment group) resulted in a rapid recovery of severe thrombocytopenia similar to the platelet count increases observed in TTP patients treated by plasma exchange. Baboons in the control group only injected with 3H9 developed early stages of TTP as previously described. Hence, inhibiting VWF-GPIb interactions is an effective way to prevent and treat the early symptoms of acquired TTP in baboons. (Blood. 2012;120(17): 3611-3614)