ALBERT

Description: Abstract 4671 TTP results from deficiency of ADAMTS13 that leads to accumulation of high molecular weight multimers (HMWM) of von Willebrand factor (VWF) and thrombosis in the microvasculature. Previous studies have shown that HMWM VWF purified from blood group O individuals were cleaved faster by ADAMTS13 compared to HMWM VWF from non-O blood group individuals. We hypothesized that blood group O patients have lower prevalence of TTP and/or less severe form of TTP. We conducted a retrospective chart review of all TTP patients treated at our institution from 1993 to 2010. The patients were identified from the review of plasmapheresis treatment records. Any patients with missing charts were excluded. The diagnosis of TTP was made on the basis of laboratory evidence of microangiopathic hemolytic anemia and thrombocytopenia and neurologic and/or renal abnormalities, following exclusion of alternative diagnoses. ADAMTS13 levels were determined by collagen binding assay in select patients. 94 patients were included in the study. The patient characteristics were as follows: 32 males versus 62 females, mean age 45.4 years. 37/94 (39%) patients had ADAMTS13 analyzed with the following results: low ADAMTS13 (21 patients), normal ADAMTS13 (15 patients), equivocal 1. Presentation laboratory investigations were available for 91 patients and were as follows: median platelet count 16×109/L (min 3, max 274) and median hemoglobin 89 g/L (min 48, max 152). At presentation, 19/91 (20.9%) patients had fever (〉38C) and 43/91 (47%) had neurological abnormalities (ranging in severity from headache to seizures and decreased level of consciousness). The blood group distribution in the TTP cohort was blood group O 41 patients, non-O 48 patients, data not available 5 patients. All patients received plasmapheresis with cryosupernatant plasma (CSP) or FFP, with some also receiving steroids and other therapies. The patients received a median of 12 (min 1, max 67) plasmapheresis treatments. Overall mortality rate was 22%. The other outcomes were as follows: death at 6 months follow-up 19%; death at 6 months or relapse 20%, death during treatment or permanent dialysis or permanent neurological disability 21%. The prevalence of blood group O versus non-O in our TTP cohort (n=89) was not significantly different from the prevalence of blood group O versus non-O in the general patient population of our institution over 2000–2010 (n=24,852; p=0.71). As illustrated below, in univariate analysis, TTP patients with blood group O did not have significantly different outcomes compared to TTP patients with non-O group blood. Outcomes Group O patients Non-group O patients p-values Death at 6 months 6/41 11/48 0.32 Death at 6 months or relapse 13/41 20/48 0.16 Death during treatment/permanent disability/permanent dialysis dependence 7/41 10/48 0.38 Conclusion: The prevalence of blood group O vs. non-O was not significantly different between the TTP cohort and our general patient population. Although there appeared to be a trend towards worse outcomes in patients with non-O blood group, there were no statistically significant differences in the outcomes in the patients with blood group O vs. non-O. The outcomes assessed in our study included death at 6 months, death at 6 months or relapse, and death during treatment/permanent neurological disability/permanent dialysis dependence. Disclosures: No relevant conflicts of interest to declare.

Description: Apoptosis, or programmed cell death, is appreciated as the main physiologic mechanism that regulates cell life-span and serves for controlled deletion of unwanted cells. Since its discovery in 1972, apoptosis was long attributed exclusively to nucleate cells. It took more than 20 years to recognize apoptosis in enucleated cells cytoplasts and anucleate platelets. During the following years, apoptosis has been demonstrated in platelets treated with natural and artificial agonists, in platelet concentrates aged during storage under standard blood banking conditions, and in animal models of suppressed thrombopoiesis and thrombocytopenia. Other studies documented that mechanical forces (shear stresses) stimulate platelet activation and signaling in the absence of exogenous chemical stimuli. We analysed whether shear stresses can trigger platelet apoptosis, a question that has not yet been studied. Using a cone-and-plate viscometer (CAP-2000, Brookfield Engineering Labs, Inc., Middleboro, MA), we exposed human platelet-rich plasma to different shear stresses, ranging from physiologic arterial and arterioles levels (10–44 dynes/cm2) to pathologic high levels (117–388 dynes/cm2) occurring in stenosed coronary, peripheral or cerebral arteries. We found that pathologic shear stresses induce not only platelet activation (P-selectin upregulation and GPIb-alpha downregulation) but also trigger apoptosis events, including mitochondrial transmembrane potential depolarization, caspase 3 activation, phosphatidylserine exposure, and platelet shrinkage and fragmentation into microparticles, whereas physiologic shear stresses are not effective. Platelets subjected to pathologic shear stresses are characterized by impaired platelet function as shown by the absence of ADP-induced platelet aggregation. Apoptosis changes were also induced by the treatment of platelets with calcium ionophore A23187 (10 μM) and thrombin (1 U/mL). Thus, in the present work, we have demonstrated that platelet apoptosis can be induced by chemical stimuli and by mechanical rheological forces (pathologic high shear stresses). Most of shear-induced activation and apoptosis events occur inside of the platelet, including translocation of CD62 from alpha-granules to the platelet surface, depolarization of mitochondrial inner membrane potential, activation of cytosolic enzyme caspase 3, and translocation of phosphatidylserine from the inner to the outer plasma membrane leaflet. These data suggest that the effects of shear stress on platelet activation and apoptosis are mediated by mechanoreceptor(s) that transmit activation and apoptosis signals to the cell interior. The platelet paradigm of apoptosis induced by chemical agonists and shear stresses suggests that apoptotic cytoplasmic machinery may function without nuclear participation.

Description: Abstract 4675 Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening form of microangiopathic hemolysis that can be associated with pregnancy. Although the association between TTP and pregnancy is well recognized, the presentation, natural history, and ideal treatment of TTP in this population remain poorly understood. St. Michael's Hospital is a regional referral centre for TTP in Ontario, Canada. We conducted a retrospective review of all cases of TTP referred to our pheresis unit between July 1993 and May 2010. Ninety-four cases were identified, of these, ten patients were pregnant or up to one week post-partum at the time of TTP diagnosis. Median age at presentation was 34 (29-36). Only one patient had been previously diagnosed with TTP and then developed a TTP relapse while pregnant. At presentation, all patients had thrombocytopenia (platelets: 〈 3 to 124 × 106/ml), anemia (hemoglobin: 60 to 99 mg/L), elevated LDH (1.3 to 12.5 × ULN), and fragmentation on blood film. ADAMTS13 was assessed in six of ten patients. It was normal in four patients, and deficient in two patients. Seven patients had neurological symptoms at presentation, one of whom suffered permanent right sided paralysis. Six patients had depressed glomerular filtration ratio (GFR) at presentation, four of these six recovered normal GFR. Hypertension was present in only one patient, and no patients had abnormal coagulation parameters at presentation. ALT was normal in six patients, mildly elevated in two patients, and unavailable in two patients. There were no patient deaths in this series. Patients were treated with a median of 17 plasma exchanges (range: 9 to 55) using either fresh frozen plasma or cryosupernatant. Eight patients achieved complete remission with plasma exchange. Two patients had refractory disease which ultimately responded to splenectomy and rituximab respectively. There were two fetal deaths, one a first trimester miscarriage, and the second an intrauterine death in the second trimester. In conclusion, in our series of pregnancy associated-TTP, there were no maternal deaths and most women responded to a short course of plasma exchange. ADAMTS13 deficiency was uncommon in our series. Nonetheless, in our series, TTP was not difficult to distinguish from preeclampsia and/or disseminated intravascular coagulation using routine laboratory parameters. Disclosures: No relevant conflicts of interest to declare.

Description: Platelets become activated during preparation and storage of platelet concentrates (PCs) for transfusion. Flow cytometric assays of platelet activation can be employed for quantifying in vitro quality of PCs. It remains, however, unclear whether the level of in vitro platelet activation in stored PCs correlates with in vivo survival of the platelets after transfusion. Platelet surface glycoprotein (GP) Ibα and P-selectin (CD62) can be involved in regulation of posttransfusion PC clearance, mediating adhesive interactions of platelets with counter-receptors on leukocytes and endothelial cells. Recently, we described a rabbit model for analyzing posttransfusion kinetics of human PCs (Leytin et al, Transfusion42:711, 2002, Transfusion43:983, 2003). In the present work, we used this validated model for studying the implication of CD62 and GPIbα expression in posttransfusion PC clearance. Platelet activation in vitro was determined by flow cytometry using anti-CD62 and anti-GPIbα antibodies. PC clearance in vivo was evaluated in rabbits with inhibited reticuloendothelial system, as measured by 0.5 hr (R0.5), 24 hr (R24) and total (R∑) platelet recoveries, and survival time (ST). Correlations were analysed between in vitro assays of platelet activation and in vivo clearance of conventional (Day 2–5) and outdated (Day 7–8) PCs stored at 22°C, and refrigerated PCs. We found that the binding of anti-CD62 antibody was significantly increased in outdated and refrigerated PCs compared to conventional PCs, reflecting an increased exposure of CD62 on the platelet surface. In contrast, binding of anti-GPIbα antibody was significantly decreased during prolonged and refrigerated PC storage. The clearance of conventional (Day 2–5) PCs from the circulation can be described by a biphasic survival curve. The first (early) phase of platelet clearance is characterized by fast (≥ 14 x 109 platelets per hour) platelet removal, whereas the second (delayed) phase has a much slower rate of platelet clearance (approximately 0.4 x 109 platelets per hour). The biphasic survival curves were also obtained for outdated and refrigerated PCs, and were employed for determining fast (R0.5), delayed (ST and R24) and overall (fast + delayed; R∑) platelet clearance in vivo. We found that when stored PCs are cold-damaged, their in vivo viability decreased significantly, in comparison to conventional PCs, as reflected by the fast, delayed and overall platelet clearances. Viability of Day 7–8 PCs is also decreased, compared to Day 2–5 PCs, but only the fast and overall platelet clearance increased significantly. Negative correlation was observed between in vitro anti-CD62-binding to platelets and their fast, but not delayed, clearance. In contrast, anti-GPIbα-binding showed positive correlations with delayed, but not fast, platelet clearance. Overall clearance correlated better with anti-GPIbα- than with anti-CD62-binding. We also demonstrated that CD62 is shed from the platelet surface after transfusion, whereas GPIbα remains unchanged on the surface of circulating platelets. The data suggest that CD62 exposure during PC storage triggers fast CD62-mediated PC clearance. However, after CD62 shedding during platelet circulation, in vitro GPIbα alterations, such as cleavage, clustering or conformation changes, may determine long-term GPIbα-mediated PC clearance.