Andrea Artoni

Caplacizumab for Acquired Thrombotic Thrombocytopenic Purpura

BACKGROUND Acquired thrombotic thrombocytopenic purpura (TTP) is caused by aggregation of platelets on ultralarge von Willebrand factor multimers. This microvascular thrombosis causes multiorgan ischemia with potentially life-threatening complications. Daily plasma exchange and immunosuppressive therapies induce remission, but mortality and morbidity due to microthrombosis remain high. METHODS Caplacizumab, an anti-von Willebrand factor humanized single-variable-domain immunoglobulin (Nanobody), inhibits the interaction between ultralarge von Willebrand factor multimers and platelets. In this phase 2, controlled study, we randomly assigned patients with acquired TTP to subcutaneous caplacizumab (10 mg daily) or placebo during plasma exchange and for 30 days afterward. The primary end point was the time to a response, defined as confirmed normalization of the platelet count. Major secondary end points included exacerbations and relapses. RESULTS Seventy-five patients underwent randomization (36 were assigned to receive caplacizumab, and 39 to receive placebo). The time to a response was significantly reduced with caplacizumab as compared with placebo (39% reduction in median time, P=0.005). Three patients in the caplacizumab group had an exacerbation, as compared with 11 patients in the placebo group. Eight patients in the caplacizumab group had a relapse in the first month after stopping the study drug, of whom 7 had ADAMTS13 activity that remained below 10%, suggesting unresolved autoimmune activity. Bleeding-related adverse events, most of which were mild to moderate in severity, were more common with caplacizumab than with placebo (54% of patients vs. 38%). The frequencies of other adverse events were similar in the two groups. Two patients in the placebo group died, as compared with none in the caplacizumab group. CONCLUSIONS Caplacizumab induced a faster resolution of the acute TTP episode than did placebo. The platelet-protective effect of caplacizumab was maintained during the treatment period. Caplacizumab was associated with an increased tendency toward bleeding, as compared with placebo. (Funded by Ablynx; ClinicalTrials.gov number, NCT01151423.).

Effects of inhalable particulate matter on blood coagulation.

See also Mannucci PM. Fine particulate: it matters. This issue, pp 659–61; Dales RE, Cakmak S, Vidal CB. Air pollution and hospitalization for venous thromboembolic disease in Chile. This issue, pp 669–74.

An intact PDZ-motif is essential for correct P2Y12 purinoceptor traffic in human platelets

The platelet P2Y(12) purinoceptor (P2Y(12)R), which plays a crucial role in hemostasis, undergoes internalization and subsequent recycling to maintain receptor responsiveness, processes that are essential for normal platelet function. Here, we observe that P2Y(12)R function is compromised after deletion or mutation of the 4 amino acids at the extreme C-terminus of this receptor (ETPM), a putative postsynaptic density 95/disc large/zonula occludens-1 (PDZ)-binding motif. In cell line models, removal of this sequence or mutation of one of its core residues (P341A), attenuates receptor internalization and receptor recycling back to the membrane, thereby blocking receptor resensitization. The physiologic significance of these findings in the regulation of platelet function is shown by identification of a patient with a heterozygous mutation in the PDZ binding sequence of their P2Y(12)R (P341A) that is associated with reduced expression of the P2Y(12)R on the cell surface. Importantly, platelets from this subject showed significantly compromised P2Y(12)R recycling, emphasizing the importance of the extreme C-terminus of this receptor to ensure correct receptor traffic.

Integrin beta3 regions controlling binding of murine mAb 7E3: implications for the mechanism of integrin alphaIIbbeta3 activation.

The integrin αIIbβ3 and αVβ3 receptors are important in a number of physiologic and pathologic phenomena, including hemostasis, thrombosis, tumor angiogenesis, and bone resorption (1, 2). The murine mAb 7E3 (3) blocks ligand binding to both αIIbβ3 and αVβ3 receptors (4, 5). Abciximab is a mouse/human chimeric Fab fragment of the mAb 7E3 that inhibits αIIbβ3-mediated platelet aggregation and is approved for human use to prevent the ischemic complications associated with percutaneous coronary interventions (6). Previous studies of 7E3 binding to cells expressing recombinant αIIbβ3 receptors demonstrated that: (i) swapping select murine for human αIIb sequences does not decrease 7E3 binding (7), (ii) removing the specificity determining loop (SDL) (K156–G189 sequence) from β3 results in loss of 7E3 binding (8), (iii) swapping the murine for human C177–C184 sequence within the SDL region in β3 results in loss of 7E3 binding (7), and (iv) swapping the murine S129–T133 sequence for the human W129–N133 sequence results in partial loss of 7E3 binding (7). The above human/mouse swapping studies identified regions within β3 that affect 7E3 binding, but the biochemical and functional properties of these chimeric receptors were not characterized. In addition, the W129–N133 region contains two amino acid differences between human β3(β3Hu) and mouse β3 (β3M) and the C177–C184 region contains three amino acid differences (Table 1). To define further the regions on αIIbβ3 that control 7E3 binding, we assessed the effects of individual amino acid substitutions on 7E3 binding to cells expressing αIIbβ3, as well as the effects of these substitutions on receptor biochemistry and function. In addition, we studied the effect of mutating a highly conserved lysine residue (K125) that appears to link the C177–C184 loop to the α-helix containing W129–N133. Table 1. Constructs for mammalian cell expression Finally, in view of our localization of the region involved in 7E3 binding to the head region of β3 adjacent to the arginine-glycine-aspartic acid (RGD) binding site, our previous studies demonstrating that 7E3 IgG (but not 7E3 Fab) binds much more rapidly to activated than unactivated platelets, (3, 9), and our recently proposed model of αIIbβ3 undergoing a change from a bent to an extended conformation upon activation, (10, 11), we also assessed the binding of 7E3 IgG and 7E3 Fab to an αIIbβ3 receptor reversibly locked in a bent conformation (10).

Metformin overdose causes platelet mitochondrial dysfunction in humans

IntroductionWe have recently demonstrated that metformin intoxication causes mitochondrial dysfunction in several porcine tissues, including platelets. The aim of the present work was to clarify whether it also causes mitochondrial dysfunction (and secondary lactate overproduction) in human platelets, in vitro and ex vivo.MethodsHuman platelets were incubated for 72 hours with saline or increasing doses of metformin (in vitro experiments). Lactate production, respiratory chain complex activities (spectrophotometry), mitochondrial membrane potential (flow-cytometry after staining with JC-1) and oxygen consumption (Clark-type electrode) were then measured. Platelets were also obtained from ten patients with lactic acidosis (arterial pH 6.97 ± 0.18 and lactate 16 ± 7 mmol/L) due to accidental metformin intoxication (serum drug level 32 ± 14 mg/L) and ten healthy volunteers of similar sex and age. Respiratory chain complex activities were measured as above (ex vivo experiments).ResultsIn vitro, metformin dose-dependently increased lactate production (P < 0.001), decreased respiratory chain complex I activity (P = 0.009), mitochondrial membrane potential (P = 0.003) and oxygen consumption (P < 0.001) of human platelets. Ex vivo, platelets taken from intoxicated patients had significantly lower complex I (P = 0.045) and complex IV (P < 0.001) activity compared to controls.ConclusionsDepending on dose, metformin can cause mitochondrial dysfunction and lactate overproduction in human platelets in vitro and, possibly, in vivo.Trial registrationNCT%2000942123.

A polymorphism in the human serotonin 5-HT2A receptor gene may protect against systemic sclerosis by reducing platelet aggregation

IntroductionPlatelet aggregation may contribute to the pathogenesis of systemic sclerosis: following activation, platelets release significant amounts of serotonin – which promotes vasoconstriction and fibrosis, and further enhances aggregation. The C+1354T polymorphism in the exonic region of the serotonin 2A receptor gene determining the His452Tyr substitution was associated with blunted intracellular responses after serotonin stimulation, and may have a role in susceptibility to scleroderma.MethodsOne hundred and fifteen consecutive systemic sclerosis patients and 140 well-matched healthy control individuals were genotyped by sequence-specific primer-PCR for the His452Tyr substitution of the serotonin 2A receptor gene, and associations were sought with scleroderma and its main clinical features. The functional relevance of the His452Tyr substitution was also assessed by evaluating the aggregation of platelet-rich plasma from His452/His452 and His452/Tyr452 healthy individuals after stimulation with adenosine diphosphate ± serotonin.ResultsThe T allele of the C+1354T polymorphism was underrepresented in scleroderma patients compared with control individuals (5.2% versus 12.4%, P < 0.001, chi-square test and 1,000-fold permutation test) and its carriage reduced the risk for systemic sclerosis (odds ratio = 0.39, 95% confidence interval = 0.19 to 0.85, P < 0.01). Platelets from His452/Tyr452 healthy subjects more weakly responded to serotonin stimulation compared with platelets from His452/His452 individuals (3.2 ± 2.6-fold versus 9.6 ± 8.6-fold increase in aggregation, P = 0.017 by Kolmogorov–Smirnov test and P = 0.003 after correction for baseline adenosine diphosphate-induced aggregation values).ConclusionThe His452Tyr substitution may influence susceptibility to systemic sclerosis by altering platelet aggregation in response to serotonin.

Integrin β3 regions controlling binding of murine mAb 7E3: Implications for the mechanism of integrin αIIbβ3 activation

The integrin αIIbβ3 and αVβ3 receptors are important in a number of physiologic and pathologic phenomena, including hemostasis, thrombosis, tumor angiogenesis, and bone resorption (1, 2). The murine mAb 7E3 (3) blocks ligand binding to both αIIbβ3 and αVβ3 receptors (4, 5). Abciximab is a mouse/human chimeric Fab fragment of the mAb 7E3 that inhibits αIIbβ3-mediated platelet aggregation and is approved for human use to prevent the ischemic complications associated with percutaneous coronary interventions (6). Previous studies of 7E3 binding to cells expressing recombinant αIIbβ3 receptors demonstrated that: (i) swapping select murine for human αIIb sequences does not decrease 7E3 binding (7), (ii) removing the specificity determining loop (SDL) (K156–G189 sequence) from β3 results in loss of 7E3 binding (8), (iii) swapping the murine for human C177–C184 sequence within the SDL region in β3 results in loss of 7E3 binding (7), and (iv) swapping the murine S129–T133 sequence for the human W129–N133 sequence results in partial loss of 7E3 binding (7). The above human/mouse swapping studies identified regions within β3 that affect 7E3 binding, but the biochemical and functional properties of these chimeric receptors were not characterized. In addition, the W129–N133 region contains two amino acid differences between human β3(β3Hu) and mouse β3 (β3M) and the C177–C184 region contains three amino acid differences (Table 1). To define further the regions on αIIbβ3 that control 7E3 binding, we assessed the effects of individual amino acid substitutions on 7E3 binding to cells expressing αIIbβ3, as well as the effects of these substitutions on receptor biochemistry and function. In addition, we studied the effect of mutating a highly conserved lysine residue (K125) that appears to link the C177–C184 loop to the α-helix containing W129–N133. Table 1. Constructs for mammalian cell expression Finally, in view of our localization of the region involved in 7E3 binding to the head region of β3 adjacent to the arginine-glycine-aspartic acid (RGD) binding site, our previous studies demonstrating that 7E3 IgG (but not 7E3 Fab) binds much more rapidly to activated than unactivated platelets, (3, 9), and our recently proposed model of αIIbβ3 undergoing a change from a bent to an extended conformation upon activation, (10, 11), we also assessed the binding of 7E3 IgG and 7E3 Fab to an αIIbβ3 receptor reversibly locked in a bent conformation (10).

Anticoagulant Treatment With Rivaroxaban in Severe Protein S Deficiency

We report a case of a 6-year-old girl with severe protein S deficiency due to a homozygous mutation and recurrent episodes of skin necrosis. She developed purpura fulminans at birth and a catheter-related venous thrombosis complicated by massive pulmonary embolism at the sixth day of life. Long-term oral anticoagulant therapy with a vitamin K-antagonist was started with a therapeutic range of the international normalized ratio of prothrombin time between 2.0 and 3.0. Unfortunately, this common range was not sufficient because recurrent episodes of warfarin-induced skin necrosis developed if the international normalized ratio was <4.0. Vitamin K antagonists decrease plasma level of vitamin K–dependent coagulation proteins, including the natural anticoagulant protein C. In our patient, the hypercoagulable state due to warfarin-induced reduction of protein C, other than severe protein S deficiency, outweighed the anticoagulant efficacy of the inhibition of procoagulant factors II, VII, IX, and X. The switch of anticoagulant therapy from warfarin to rivaroxaban, a direct inhibitor of activated factor X that does not inhibit other vitamin K–dependent proteins, resulted in the disappearance of skin necrosis at 1 year of follow-up. Rivaroxaban may be considered as a valid anticoagulant alternative in patients with severe inherited protein S deficiency and warfarin-induced skin necrosis.

Measurement and prevalence of circulating ADAMTS13-specific immune complexes in autoimmune thrombotic thrombocytopenic purpura

The formation of ADAMTS13‐specific circulating immune complexes (CICs) may be a pathophysiologic mechanism in autoimmune thrombotic thrombocytopenic purpura (TTP), but has not been systematically investigated.

Genetic characterization of patients with Bernard-Soulier syndrome and their relatives from Southern Iran

Bernard-Soulier syndrome (BSS) is a rare recessively inherited bleeding disorder caused by the deficiency of the platelet glycoprotein (Gp) complex Ib/IX/V that is the von Willebrand factor receptor on platelets. In patients suffering from BSS platelet adhesion is typically impaired, while platelet aggregation is normal; macrothrombocytopenia is a common feature. In this study three different families from Southern Iran were investigated. GpIb/IX/V platelet expression as detected by flow cytometry was less than 2% of normal in six cases and 12% in the remaining one. Platelet count was 35 000 platelets/microliter and iron deficiency anemia was common. All patients suffered from mucocutaneous bleeding at presentation and were born from consanguineous marriages. Genetic analysis demonstrated the presence of the same GpIX Phe55Ser missense mutation in two families and of a single base insertion (GP1BA C3221 ins), a never described mutation causing a frameshift in the GpIbα gene, in the third family. Among the family members studied several heterozygotes were identified. None of them, with one exception, had macrothrombocytopenia. In one family a slight reduction of GpIb/IX/V expression was observed.