Prothrombotic Disorder Research Articles

HMGB1 As a Novel Platelet Agonist That Acts Synergistically with ADP to Activate Platelets in Sickle Cell Disease

Background: Sickle cell disease (SCD) is a proinflammatory and prothrombotic disorder that exhibits increased platelet activation. High mobility group box 1 (HMGB1) is a nuclear protein that can mediate inflammation when released from inflammatory or ischemic cells. HMGB1 is increased in many inflammatory disease states including SCD. Recent data suggests HMGB1 activates platelets and may work synergistically with potent platelet agonists such as collagen and thrombin, but little is known regarding HMGB1-platelet interactions in combination with weaker agonists like ADP, or in isolated platelets. Moreover, the effect of HMGB1 on platelet activation has not been evaluated in SCD. We hypothesized that the in vitro addition of low-dose recombinant HMGBI (rHMBG1) to isolated platelets will lower the threshold dose of physiologic agonists required to achieve platelet activation, and that this effect is exaggerated in SCD.Methods: Platelets were isolated from healthy controls (n=4) and patients with hemoglobin SS disease (SCD; n=5). The level of platelet activation was assessed after treatment with ADP at concentrations of 0 μM, 0.5 μM, 2 μM, and 5 μM with the addition of either low-dose rHMGB1 (10 μg/mL) or the same volume of vehicle. Percent platelet activation was measured via flow cytometry using PE antibody to GPIIb (CD41) to select for platelets, and PAC1 to detect the activation-dependent conformational change in integrin αIIbβ3 (GP IIb-IIIa). Platelet activation was interpreted as percent of platelets that bound PAC1. Data was analyzed using FlowJo software and nonparametric statistical tests.Results: Mean baseline platelet activation was 1.5% (range 0.4-3.3%) for control platelets and 7.3% (1.4-17.7%) for SCD platelets (p=0.19). In the SCD group, the addition of low-dose rHMGB1 (10 μg/mL) increased the mean percent of activated platelets from 7.3% to 26.5% (10.9-43%) (p=0.01). In comparison, mean activation of control platelets increased from only 1.5% to 19.5% (8.3-42.7%) after addition of rHMGB1 (p=0.12).Having illustrated that rHMGB1 can activate washed SCD platelets, we then compared the synergistic effect of rHMGB1 with ADP. There was increased platelet activation observed when ADP was added to rHMGB1 in SCD platelets: ADP 0.5 μM increased mean platelet activation from 13.8% (range 0.3-25.3%) to 54.4% (6.7-84.9%) with the addition of rHMGB1 (p=0.02); ADP 2 μM increased platelet activation from 14.1% (2.8-23.8%) to 56.2% (22.2-88.6%) with rHMGB1 (p=0.006); and ADP 5 μM increased platelet activation from 21.4% (2.5-30.1%) to 65.3% (31.7-85.9%) after adding rHMGB1 (p=0.004) (Fig 1; * and **, diamonds vs stars). We did not find a similar statistically significant synergistic effect in the control samples treated with ADP compared to combined HMGB1 + ADP, except at ADP dose 2 μM where platelet activation increased from 14.94% (4.6-28.6%) to 39.1% (19.5-56.0%) after the addition of rHMGB1 (p=0.04; Fig 1; #, square vs circle).Activation of platelets with just ADP was not different comparing control with SCD platelets (Fig 1; circles vs stars). Similarly, activation of platelets with both ADP and rHMGB1 was not significantly different comparing control with SCD platelets except for a trend at 0.5 μM ADP + rHMGB1 10 μg/mL with 19.29% (6.6-38.7) in controls vs 54.44% (6.7-84.9) in the SCD group (p=0.07) (Fig 1; diamonds vs squares).Summary: We found that rHMGB1 acts both independently and synergistically with ADP to increase platelet activation in SCD platelets. In our small cohort, SCD platelets had increased responsiveness to low dose-rHMGB1 compared to control platelets. Moreover, combining rHMGB1 with ADP greatly enhanced platelet activation in SCD but not control platelets. Our data suggest that SCD platelets are sensitized to HMGB1 in the presence of weaker agonists such as ADP. This heightened responsiveness of SCD platelets to HMGB1 may explain the enhanced platelet activation and inflammation associated with SCD in vivo. With further study, HMGB1 could be a target of clinical drug-directed therapy in SCD patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Venous Thromboembolism in Children 0-18 Years — a Regional Population-Based Study from Sweden

Introduction: Venous thromboembolism (VTE) is a rare complication in childhood. Pediatric VTE is an important and increasingly frequent clinical challenge likely due to increased detection and advanced medical interventions leading to improved survival of previously fatal conditions.Objective: The principal aim of this population based study was to describe the incidence, age distribution, type/location of VTE, and acquired and genetic pro-thrombotic risk factors of VTE and recurrence of VTE in children 0-18 years.Material and Methods: The Regional Ethical Review Board in Lund approved the study. We conducted a retrospective regional study of all consecutive ICD-10 codes of VTE in children 0-18 years over a 15-year period (January 1, 2000, to December 31, 2015) in a regional catchment area of southern Sweden using an electronic diagnosis registry. Eligible subjects were defined as children under the age of 18 who presented with VTE and had imaging evidence of thrombosis. Of the 174 patients diagnosed with VTEs, 164 fulfilled the study group criteria. Data regarding subject demographics and medical history (central venous catheter, cancer, congenital heart disease, history of VTE, current infection, etc.), location of VTE and imaging method (upper, lower extremities, pulmonary embolism, renal, cardiac, cerebral sinus venous thrombosis (CSVT), etc.), coagulation studies at primary investigation which included in all cases evaluation of at least plasma concentrations of protein C, protein S, antithrombin, resistance to activated protein C and the genotypes FV-G1691A and FII-G20210A. In addition, plasma values for coagulation factors VIII and XI, D-dimer, PK-INR, and cardiolipin antibodies were analyzed.Results: The incidence of VTE in children in the investigated region of Sweden was found to be 0.8 per 10,000 children. Of the study group with confirmed VTE (n=164), 73/164 (45%) were males and 91/164(55%) females, with bimodal age distribution at diagnosis, 25 (15%) < 1 month, 139 (85%) >1 month-18 years. Of the children, 143/164 (87%) had DVT (deep venous thrombosis), 21/164 (13%) had PE (pulmonary embolism) and 5/164 (3%) had both DVT and PE. Of 143 patients with DVT, 50 (30%) had lower extremity DVT, 46 (28%) had upper extremity DVT and 34 (20.7%) CSVT and the remaining 13 various locations. 79/164 (59%) had acquired potential risk factors, 11/164 (11%) had genetic risk factors, 34/164 (21%) had both genetic and acquired risk factors, and 22/164 (13%) had no identified risk factors. The most frequent acquired risk factors in the cohort were the use of hormonal therapy (34%), concomitant malignancy (21%), infection at the time of thrombosis (19%) or a CVL (central venous line) (15%). Genetic thrombophilia risk factors were found in 45/164 (27.5%), the most common were Factor V Leiden (FVL) in heterozygous form in 35 (21%), FII mutation (heterozygous) in 4 (2%) and double heterozygosity for FVL and FII mutation found in 2 (1%). Plasma deficiency of Protein S was found in 5, Protein C deficiency in 6 and Antithrombin deficiency in 1 patient (who had 3 episodes of VTE). Recurrent VTE was documented in 9 (5%), of which 5 had a congenital pro-thrombotic disorder (i.e. FVL mutation (n=3), antithrombin deficiency (n=1) and a protein S deficiency (n=1). Two out of the nine with recurrent VTE had neither a genetic nor an acquired identified risk factor. Six out of a total of 45 (13.3%) with genetic risk factors had a recurrent VTE. No common acquired pro-thrombotic risk factor was found in the group with recurrent VTE.Conclusion: The incidence, age-distribution, locations and underlying disorders agree with published findings in pediatric populations. In our study, 87% of the children with VTE had either an identifiable acquired or genetic risk factor or a combination of both. Of those with a genetic risk factor, 13% had a recurrent VTE during the study period which indicates an even higher cumulative risk during childhood which emphasizes the need to consider prophylaxis in situations with increased risk of VTE. However, of those with recurrent VTE, no frequent acquired risk factor was identified. DisclosuresNo relevant conflicts of interest to declare.

Heparin‐induced thrombocytopenia‐associated thrombosis: from arterial to venous to venous limb gangrene

Heparin-induced thrombocytopenia (HIT) is an acquired immune-mediated hypercoagulability state that is strongly associated with thrombosis. During the 1970s and 1980s, the prevailing concept was that HIT was associated only with arterial thrombosis, through its unique pathogenesis via heparin-dependent, platelet-activating IgG antibodies. However, in 1990, when I began to encounter HIT in my clinical practice, I found that most such patients developed symptomatic venous thrombosis. This historical sketch summarizes some of the research that challenged the dogma of HIT being a mainly arterial prothrombotic disorder. Two studies - one a substudy of a randomized trial of post-orthopedic surgery thromboprophylaxis, and the second a retrospective five-hospital analysis of consecutive patients with positive test results for HIT antibodies - showed a marked predominance of venous over arterial thrombosis complicating HIT (~4:1). By the end of the 1990s, an even more dramatic manifestation of HIT-associated venous thrombosis was recognized: venous limb gangrene. Here, ischemic limb necrosis occurs despite palpable arterial pulses, as a result of both macrovascular and microvascular venous thrombosis. The surprising explanation was natural anticoagulant impairment (severe depletion of proteinC, a vitaminK-dependent anticoagulant) resulting from treatment of HIT-associated deep vein thrombosis with warfarin (vitaminK antagonist). These insights from HIT research helped to elucidate the pathogenesis of ischemic limb losses in other intense non-HIT hypercoagulability states, including warfarin-associated venous limb gangrene complicating cancer-associated hypercoagulability, and symmetrical peripheral gangrene complicating disseminated intravascular coagulation of critical illness, in which proximate 'shock liver' helps to explain the profound failure of natural anticoagulant systems (proteinC; antithrombin) in predisposing to peripheral limb microthrombosis in circulatory shock.

Study of clinical utility of antibodies to phosphatidylserine/prothrombin complex in Asian-Indian patients with suspected APS.

Antiphospholipid syndrome (APS) is the most common acquired pro-thrombotic disorder, also associated with obstetric complications. Phosphatidylserine/Prothrombin complex antibody (aPSPT) though associated with various APS manifestations, is not included in the revised Sapporo Criteria. To study the prevalence of aPSPT in Asian-Indian patients with suspected APS and compare its performance with the criteria anti-phospholipid antibodies (APLs). Electronic charts of 372 individuals whose sera was tested for aPSPT in suspected APS between June 2014 and May 2016 were retrieved and analyzed. aPSPT was assayed by ELISA. aPSPT tested individuals were categorized into cases-seropositive and seronegative APS (SNAPS) and controls. aPSPT was positive in 24/58 (41.3%) cases and 17/314 (5.4%) controls (p < 0.001). aPSPT positivity was seen in 44.5%, 38.7%, and 58.4% in primary, secondary and SNAPS patients respectively. aPSPT had the best performance among all APLs, in obstetric APS with 31% sensitivity, 97.7% specificity, and an odds ratio of 18.8. It showed 41.4% sensitivity, 94.6% specificity for the classification/diagnosis of primary APS and 38.7% sensitivity, 91.5% specificity for secondary APS. Addition of aPSPT to current APS criteria to SNAPS patients led to reclassification of additional 12.1% patients as APS overall and 42.8% in obstetric APS category. In Asian-Indian patients with suspected APS, aPSPT outperformed all classical APLs in diagnosis/classification of obstetric APS and both isotypes of beta 2-glycoprotein-I antibodies in diagnosis/classification of APS. aPSPT could reclassify additional 12.1 and 42.8% patients as APS overall and obstetric APS respectively, over and above the cases satisfying revised Sapporo criteria.

Dynamic intercellular redistribution of HIT antigen modulates heparin-induced thrombocytopenia

AbstractHeparin-induced thrombocytopenia (HIT) is a prothrombotic disorder initiated by antibodies to platelet factor 4 (PF4)/heparin complexes. PF4 released from platelets binds to surface glycosaminoglycans on hematopoietic and vascular cells that are heterogenous in composition and differ in affinity for PF4. PF4 binds to monocytes with higher affinity than to platelets, and depletion of monocytes exacerbates thrombocytopenia in a murine HIT model. Here we show that the expression of PF4 on platelets and development of thrombocytopenia are modulated by the (re)distribution of PF4 among hematopoietic and endothelial cell surfaces. Binding of PF4 to platelets in whole blood in vitro varies inversely with the white cell count, likely because of the greater affinity of monocytes for PF4. In mice, monocyte depletion increased binding of PF4 to platelets by two- to three-fold. Induction of HIT in mice caused a transient >80-fold increase in binding of HIT antibody to monocytes vs 3.5-fold increase to platelets and rapid transient monocytopenia. Normalization of monocyte counts preceded the return in platelet counts. Exposure of blood to endothelial cells also depletes PF4 from platelet surfaces. These studies demonstrate a dynamic interchange of surface-bound PF4 among hematopoetic and vascular cells that may limit thrombocytopenia at the expense of promoting prothrombotic processes in HIT.

Pregnancy-triggered triple autoimmunity (Hashimoto’s thyroiditis, antiphospholipid syndrome and systemic lupus erythematosus)

Introduction: We describe a case of 22 year old female with her first pregnancy triggered Hashimoto’s thyroiditis (HT), Antiphospholipid Syndrome (APS) and Systemic Lupus Erythematosus (SLE). Case Report: A 22 year old female was diagnosed with HT on levothyroxine during the early first trimester. During 21 weeks of gestation patient has intrauterine fetal demise and underwent medical abortion. SLE work up including antinuclear antibody and anti-double stranded DNA were positive. She underwent kidney biopsy, which revealed membranous and mesangial proliferative lupus nephritis. Diagnosis of SLE and APS was made. Treatment with anticoagulation therapy was started. SLE therapy was initiated with prednisone, mycophenolate mofetil and hydroxychloroquine with complete resolution of symptoms.We report a 23-year-old gravida in her first pregnancy, suffering from MGN and severe nephrotic syndrome, complicated by APLA syndrome. The patient was treated with enoxaparin, aspirin azathioprine, and Prednisone for a short time, in addition to furosemide and albumin intravenously. She was delivered at 30 weeks due to deteriorating maternal and foetal conditions. Discussion: APS is a prothrombotic disorder with various manifestations, most commonly venous and arterial thromboembolism and recurrent pregnancy loss. Pregnancy may trigger an underlying APS, which may well be the causative for the miscarriage. New onset SLE during pregnancy is rare. However, in our case, the anemia, thrombocytopenia, and proteinuria led us to the correct diagnosis of SLE. HT is associated with higher rates of infertility and early miscarriages, due to the associated hormonal changes and instability. However, the association of APS and HT is not well recognized in pregnant women. Conclusion: We present here a challenging case of new-onset triple autoimmune disorders trigged by pregnancy. Clinicians should be aware of this association and initiate early autoimmune work up for SLE and APS in patients with new onset of HT during pregnancy. A successful neonatal and maternal outcome was achieved in this case. The patient's history revealed thrombocytopenia and APLA syndrome and continues to be treated chronically with enoxaparin. Kidney biopsy performed after delivery showed membranous MGN stage II-III. Herein, we present a case of successful pregnancy and foetal outcome in a young woman with APLA syndrome and MN. Keywords: Pregnancy triggered Hashimoto’s thyroiditis, Antiphospholipid Syndrome and Systemic Lupus Erythematosus

Pregnancy-triggered triple autoimmunity (Hashimoto’s thyroiditis, antiphospholipid syndrome and systemic lupus erythematosus)

Introduction: We describe a case of 22 year old female with her first pregnancy triggered Hashimoto’s thyroiditis (HT), Antiphospholipid Syndrome (APS) and Systemic Lupus Erythematosus (SLE). Case Report: A 22 year old female was diagnosed with HT on levothyroxine during the early first trimester. During 21 weeks of gestation patient has intrauterine fetal demise and underwent medical abortion. SLE work up including antinuclear antibody and anti-double stranded DNA were positive. She underwent kidney biopsy, which revealed membranous and mesangial proliferative lupus nephritis. Diagnosis of SLE and APS was made. Treatment with anticoagulation therapy was started. SLE therapy was initiated with prednisone, mycophenolate mofetil and hydroxychloroquine with complete resolution of symptoms. We report a 23-year-old gravida in her first pregnancy, suffering from MGN and severe nephrotic syndrome, complicated by APLA syndrome. The patient was treated with enoxaparin, aspirin azathioprine, and Prednisone for a short time, in addition to furosemide and albumin intravenously. She was delivered at 30 weeks due to deteriorating maternal and foetal conditions. Discussion: APS is a prothrombotic disorder with various manifestations, most commonly venous and arterial thromboembolism and recurrent pregnancy loss. Pregnancy may trigger an underlying APS, which may well be the causative for the miscarriage. New onset SLE during pregnancy is rare. However, in our case, the anemia, thrombocytopenia, and proteinuria led us to the correct diagnosis of SLE. HT is associated with higher rates of infertility and early miscarriages, due to the associated hormonal changes and instability. However, the association of APS and HT is not well recognized in pregnant women. Conclusion: We present here a challenging case of new-onset triple autoimmune disorders trigged by pregnancy. Clinicians should be aware of this association and initiate early autoimmune work up for SLE and APS in patients with new onset of HT during pregnancy. A successful neonatal and maternal outcome was achieved in this case. The patient's history revealed thrombocytopenia and APLA syndrome and continues to be treated chronically with enoxaparin. Kidney biopsy performed after delivery showed membranous MGN stage II-III. Herein, we present a case of successful pregnancy and foetal outcome in a young woman with APLA syndrome and MN. Keywords: Pregnancy triggered Hashimoto’s thyroiditis, Antiphospholipid Syndrome and Systemic Lupus Erythematosus

Hughes syndrome antiphospholipid syndrome

An increasingly important link between aPL antibodies and a clinical syndrome is becoming recognized worldwide. This syndrome, known as the antiphospholipid syndrome (APS) or Hughes syndrome, is a prothrombotic disorder leading to both arterial and venous thrombosis and, in pregnancy, recurrent abortion and pregnancy loss (Figure 13.1).

Surgical Interventions for Organ and Limb Ischemia Associated With Primary and Secondary Antiphospholipid Antibody Syndrome With Arterial Involvement.

The association of antiphospholipid antibody syndrome (APS) and hypercoagulability is well known. Arterial compromise leading to ischemia of organs and/or limbs in patients with APS is uncommon, frequently unrecognized, and rarely described. We evaluated our institutional experience. Retrospective review was conducted. From August 2007 to September 2016, 807 patients with diagnosis of APS were managed in our Institution. Patients with primary and secondary APS who required interventions were examined. Demographics, comorbidities, manifestations, procedures, complications, and other factors affecting outcomes were recorded. Fourteen patients (mean age 35 years old, standard deviation ±14) were evaluated and treated by our service. Six (43%) of them had primary APS and 8 (57%) had secondary APS; 11 (79%) were female. Two (14%) experienced distal aorta and iliac arteries involvement, 3 (21%) visceral vessels disease, 2 (14%) in upper and 7 (50%) in the lower extremity vasculatures. Thirteen (93%) patients underwent direct open revascularization and 1 with hand ischemia (Raynaud disease) underwent sympathectomy. During the mean follow-up period of 48 months, reinterventions included a revision of the proximal anastomosis of an aortobifemoral bypass graft, 1 (7%) abdominal exploration for bleeding, 1 (7%) graft thrombectomy, and 4 (29%) amputations (2 below the knee, 1 above the knee, and 1 transmetatarsal). One (7%) death occurred secondary to sepsis in a patient who had acute mesenteric ischemia. Significant differences in clinical manifestations and outcomes were not observed among patients with primary and secondary APS. All patients remained on systemic anticoagulation. APS is a prothrombotic disorder that may lead to arterial involvement with less frequency than the venous circulation but has significant morbidity and limb loss rate. Arterial reconstruction seems feasible in an attempt to salvage organs and limbs; however, research is necessary to establish the optimal anticoagulation regime and long-term management following surgical interventions.

Heparin-Induced Thrombocytopenia in Cardiac Surgery Patients.

AbstractHeparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by platelet-activating antibodies that target complexes of platelet factor 4 (PF4) and heparin. Because nearly all patients undergoing cardiopulmonary bypass (CPB) are exposed to heparin and experience a postoperative platelet count fall, it is not surprising that HIT is commonly suspected in this population. However, the incidence of HIT in cardiac surgery patients who receive intraoperative and postoperative unfractionated heparin is much lower than the incidence of suspected HIT, being approximately 1 to 2%. Clinical diagnosis may therefore be particularly challenging because of the need to distinguish the common platelet count fall associated with CPB and surgery from the much less common platelet count fall associated with HIT. A biphasic platelet count pattern is characteristic of HIT in this setting. Laboratory diagnosis is also difficult because of the high frequency of anti-PF4/heparin antibody seropositivity after cardiac surgery in patients without HIT. A unique aspect of management in the cardiac surgery setting is selecting an anticoagulant for intraoperative use in patients with a history of HIT who require cardiac surgery. In this article, we review the epidemiology, clinical diagnosis, and laboratory diagnosis of HIT in cardiac surgery patients and present a conceptual framework for selecting intraoperative anticoagulation in patients with a history of HIT.

Unfractionated heparin versus low molecular weight heparins for avoiding heparin-induced thrombocytopenia in postoperative patients.

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction presenting as a prothrombotic disorder related to antibody-mediated platelet activation. It is a paradoxical immune reaction resulting in thrombin generation in vivo, which leads to a hypercoagulable state and the potential to initiate venous or arterial thrombosis. A number of factors are thought to influence the incidence of HIT including the type and preparation of heparin (unfractionated heparin (UFH) or low molecular weight heparin (LMWH)) and the heparin-exposed patient population, with the postoperative patient population at higher risk.Although LMWH has largely replaced UFH as a front-line therapy, there is evidence supporting a lack of superiority of LMWH compared with UFH regarding prevention of deep vein thrombosis and pulmonary embolism following surgery, and similar frequencies of bleeding have been described with LMWH and UFH. The decision as to which of these two preparations of heparin to use may thus be influenced by harmful effects such as HIT. We therefore sought to determine the relative impact of UFH and LMWH on HIT in postoperative patients receiving thromboembolism prophylaxis. This is an update of a review first published in 2012. The objective of this review was to compare the incidence of heparin-induced thrombocytopenia (HIT) and HIT complicated by venous thromboembolism in postoperative patients exposed to unfractionated heparin (UFH) versus low molecular weight heparin (LMWH). For this update, the Cochrane Vascular Information Specialist searched the Specialised Register (May 2016), CENTRAL (2016, Issue 4) and trials registries. The authors searched Lilacs (June 2016) and additional trials were sought from reference lists of relevant publications. We included randomised controlled trials (RCTs) in which participants were postoperative patients allocated to receive prophylaxis with UFH or LMWH, in a blinded or unblinded fashion. Studies were excluded if they did not use the accepted definition of HIT. This was defined as a relative reduction in the platelet count of 50% or greater from the postoperative peak (even if the platelet count at its lowest remained greater than 150 x 109/L) occurring within five to 14 days after the surgery, with or without a thrombotic event occurring in this timeframe. Additionally, we required circulating antibodies associated with the syndrome to have been investigated through laboratory assays. Two review authors independently extracted data and assessed the risk of bias. Disagreements were resolved by consensus with participation of a third author. In this update, we included three trials involving 1398 postoperative participants. Participants were submitted to general surgical procedures, minor and major, and the minimum mean age was 49 years. Pooled analysis showed a significant reduction in the risk of HIT with LMWH compared with UFH (risk ratio (RR) 0.23, 95% confidence interval (CI) 0.07 to 0.73); low-quality evidence. The number needed to treat for an additional beneficial outcome (NNTB) was 59. The risk of HIT was consistently reduced comparing participants undergoing major surgical procedures exposed to LMWH or UFH (RR 0.22, 95% CI 0.06 to 0.75); low-quality evidence. The occurrence of HIT complicated by venous thromboembolism was significantly lower in participants receiving LMWH compared with UFH (RR 0.22, 95% CI 0.06 to 0.84); low-quality evidence. The NNTB was 75. Arterial thrombosis occurred in only one participant who received UFH. There were no amputations or deaths documented. Although limited evidence is available, it appears that HIT induced by both types of heparins is common in people undergoing major surgical procedures (incidence greater than 1% and less than 10%). This updated review demonstrated low-quality evidence of a lower incidence of HIT, and HIT complicated by venous thromboembolism, in postoperative patients undergoing thromboprophylaxis with LMWH compared with UFH. Similarily, the risk of HIT in people undergoing major surgical procedures was lower when treated with LMWH compared to UFH (low-quality evidence). The quality of the evidence was downgraded due to concerns about the risk of bias in the included studies and imprecision of the study results. These findings may support current clinical use of LMWH over UFH as front-line heparin therapy. However, our conclusions are limited and there was an unexpected paucity of RCTs including HIT as an outcome. To address the scarcity of clinically-relevant information on HIT, HIT must be included as a core harmful outcome in future RCTs of heparin.

Endothelial antigen assembly leads to thrombotic complications in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder initiated by antibodies against complexes between human platelet factor 4 (hPF4) and heparin. A better understanding of the events that initiate the prothrombotic state may improve approaches to antithrombotic management. Here, we visualized thrombus formation in an in vivo murine model and an endothelialized microfluidic system that simulate the pathogenesis of HIT. hPF4 released from platelets predominantly bound to peri-injury endothelium and formed HIT antigenic complexes that were dissociated by heparin. In mice expressing both hPF4+ and human platelet IgG Fc receptor IIA (FcγRIIA), infusion of the HIT-like monoclonal antibody KKO increased fibrin and platelet deposition at sites of injury, followed immediately by antigen formation on proximate endothelial cells. After a few minutes, HIT antigen was detected within the thrombus itself at the interface between the platelet core and the surrounding shell. We observed similar results in the humanized, endothelialized microfluidic system. hPF4 and KKO selectively bound to photochemically injured endothelium at sites where surface glycocalyx was reduced. These studies support the concept that the perithrombus endothelium is the predominant site of HIT antigen assembly. This suggests that disrupting antigen formation along the endothelium or protecting the endothelium may provide a therapeutic opportunity to prevent thrombotic complications of HIT, while sparing systemic hemostatic pathways.

Splanchnic vein thrombosis in myeloproliferative neoplasms: pathophysiology and molecular mechanisms of disease.

Myeloproliferative neoplasms (MPNs) are the most common underlying prothrombotic disorder found in patients with splanchnic vein thrombosis (SVT). Clinical risk factors for MPN-associated SVTs include younger age, female sex, concomitant hypercoagulable disorders, and the JAK2 V617F mutation. These risk factors are distinct from those associated with arterial or deep venous thrombosis (DVT) in MPN patients, suggesting disparate disease mechanisms. The pathophysiology of SVT is thought to derive from local interactions between activated blood cells and the unique splanchnic endothelial environment. Other mutations commonly found in MPNs, including CALR and MPL, are rare in MPN-associated SVT. The purpose of this article is to review the clinical and molecular risk factors for MPN-associated SVT, with particular focus on the possible mechanisms of SVT formation in MPN patients.

Heparin-induced thrombocytopenia: research and clinical updates.

Heparin-induced thrombocytopenia (HIT) remains an important diagnosis to consider in hospitalized patients developing thrombocytopenia. HIT is an immune-mediated prothrombotic disorder caused by antibodies to platelet factor 4 (PF4) and heparin. Recent basic scientific studies have advanced our understanding of disease pathogenesis through studies of the PF4/heparin structure, immune mechanisms, and cellular basis of thrombosis. Clinical advances have also occurred in areas of HIT prevention, description of disease variants, and diagnostic strategies. Emerging anticoagulants with the potential to change HIT treatment are evolving, although with limited data. This review will provide a current perspective on HIT pathogenesis, disease features, diagnostic strategies, and role of emerging therapies for the management of HIT.

Thrombocytopenia and Microangiopathic Hemolytic Anemia Precipitated By Acute Pancreatitis: A Single Center Experience of Five Cases

IntroductionThrombotic thrombocytopenic purpura (TTP) is a prothrombotic disorder characterized by microangiopathic hemolytic anemia (MAHA) and thrombocytopenia. It has been associated with a deficiency or dysfunction of ADAMTS13, an enzyme responsible for cleaving ultra-large von Willebrand factor multimers, thus preventing spontaneous platelet adhesion and aggregation. In its absence, platelet aggregates accumulate in the microvasculature causing neurological symptoms, cardiac ischemia and renal dysfunction. Most cases in adults are idiopathic, and associated with severe ADAMTS13 deficiency (<10%) and anti-ADAMTS13 antibodies. Acquired TTP due to various conditions, such as HIV, drugs, malignancy, collagen disease, bone marrow transplant and acute pancreatitis (AP), have also been described. Some cases of these secondary TTP may be associated with no to moderate ADAMTS13 deficiency and are referred to as thrombotic microangiopathies (TMAs). If plasma exchange (PLEX) has been the treatment of choice for acquired TTP, it has not been shown to be very effective for TMAs. AP-associated MAHA and thrombocytopenia (MAHA-T) might be one exception.MethodsTo better understand the relationship between AP and MAHA-T and to evaluate its response to PLEX, we retrospectively reviewed all cases of suspected TTP/ MAHA-T treated by PLEX in the context of AP in our institution from 2005 to 2015. For the purpose of this report, we preferred to use the term MAHA-T to describe this phenomenon since TMA usually refers to a pathologic diagnosis. Finally, a literature review of published cases was also performed.ResultsFive patients were treated with PLEX for suspicion of TTP in the context of AP between January 1st, 2005 and December 31st, 2015 at our centre. Median age was 36 (31-60). Etiologies for pancreatitis were alcohol (3/5) or idiopathic (2/5). Peak lipase level ranged between 426 and 5853 U/L. One patient had 3 episodes of MAHA-T following AP, always in the context of alcohol abuse. Two patients had evidence of MAHA-T on admission, while the remaining 3 patients had an unremarkable CBC at presentation followed by an abrupt drop in platelet count (nadir, 9-23 x 109/L). All patients showed simultaneously signs of MAHA (Hb nadir between 47 and 93 g/L) with fragmentation on the blood smear and abnormal hemolytic parameters: LD level 1104-8097 U/L, bilirubin 36-176 umol/L, reticulocyte count 130-541 x109/L and undetectable haptoglobin. The median time from AP presentation to the development of laboratory or clinical features of MAHA-T in these cases was 2 days (1-3). Acute kidney injury was present in all cases with creatinine levels ranging between 118-906 umol/L. One patient required hemodialysis. Other observed end organ damage included neurologic symptoms (3/5 patients), elevated troponin (5/5 patients) and transaminitis (4/5). One patient experienced a cardiac arrest. All patients had ADAMTS13 activity measured. One patient had severe deficiency on two separate episodes (ADAMTS13 was <1% and 6% respectively). Three other patients had normal ADAMTS13 activity (average 69%) and one had a moderate deficiency by a qualitative assay. In 4 patients, where tests were performed, there was significant heterogeneity in complement protein and function studies between patients ranging from normal to depressed C3 and C4 and normal to elevated CH50. Complement genetics screen was performed in 3 cases and was normal in all 3 cases. PLEX was initiated in all patients and remission (platelet count over 150 x 109/L) was seen for all after a median of 10 daily treatments (2-12). High dose steroids were used in 4 patients. All patients survived to discharge and completely recovered their kidney function.ConclusionTTP/MAHA-T precipitated by AP is increasingly recognized and more than 40 cases have been described in the literature. Similar to our findings, time to MAHA-T occurrence is generally reported within the first 4 days after admission for AP, often when markers for pancreatitis are improving, and most patients do not have severe ADAMTS13 deficiency. While many have looked at ADAMTS13 level, to our knowledge, none have yet reported thorough complement studies and genetics. The 3 cases described herein are then the first cases suggesting this is not a complement mediated disorder. Finally, our review and cases confirm that PLEX is effective and prognosis is good in almost all cases. DisclosuresPavenski:Alexion Pharmaceuticals Inc.: Honoraria, Other: attended an entity's advisory boards.

The antigenic complex in HIT binds to B cells via complement and complement receptor 2 (CD21)

AbstractHeparin-induced thrombocytopenia is a prothrombotic disorder caused by antibodies to platelet factor 4 (PF4)/heparin complexes. The mechanism that incites such prevalent anti-PF4/heparin antibody production in more than 50% of patients exposed to heparin in some clinical settings is poorly understood. To investigate early events associated with antigen exposure, we first examined the interaction of PF4/heparin complexes with cells circulating in whole blood. In healthy donors, PF4/heparin complexes bind preferentially to B cells (>90% of B cells bind to PF4/heparin in vitro) relative to neutrophils, monocytes, or T cells. Binding of PF4 to B cells is heparin dependent, and PF4/heparin complexes are found on circulating B cells from some, but not all, patients receiving heparin. Given the high proportion of B cells that bind PF4/heparin, we investigated complement as a mechanism for noncognate antigen recognition. Complement is activated by PF4/heparin complexes, co-localizes with antigen on B cells from healthy donors, and is present on antigen-positive B cells in patients receiving heparin. Binding of PF4/heparin complexes to B cells is mediated through the interaction between complement and complement receptor 2 (CR2 [CD21]). To the best of our knowledge, these are the first studies to demonstrate complement activation by PF4/heparin complexes, opsonization of PF4/heparin to B cells via CD21, and the presence of complement activation fragments on circulating B cells in some patients receiving heparin. Given the critical contribution of complement to humoral immunity, our observations provide new mechanistic insights into the immunogenicity of PF4/heparin complexes.

Aktuelle Aspekte der heparininduzierten Thrombozytopenie

Heparin-induced thrombocytopenia (HIT) is a drug-mediated, prothrombotic disorder caused by immunization against platelet factor 4 (PF4)/heparin complexes. A subset of anti-PF4 /heparin antibodies is capable of intravascular platelet activation by cross-linking Fcγ receptor IIA leading to platelet count decrease and/or thrombosis. HIT can be potentially associated with devastating complications such as life-threatening thrombosis. Therefore, if HIT is strongly suspected, all sources of heparin must be stopped and an alternative non-heparin anticoagulant should be started to prevent new thromboembolic complications. However, the diagnosis of HIT based on clinical information is often difficult. This review highlights the pathophysiology of HIT, emphasizing characteristic clinical features and the role of laboratory assays in the diagnosis of HIT. In addition, a summary of current therapeutic options for patients with HIT will be provided.

Management of heparin-induced thrombocytopenia.

The purpose of this review is to summarize recent findings on heparin-induced thrombocytopenia (HIT), a prothrombotic disorder caused by platelet-activating IgG targeting platelet factor 4 (PF4)/polyanion complexes. HIT can explain unusual clinical events, including adrenal hemorrhages, arterial/intracardiac thrombosis, skin necrosis, anaphylactoid reactions, and disseminated intravascular coagulation. Sometimes, HIT begins/worsens after stopping heparin ('delayed-onset' HIT). Various HIT-mimicking disorders are recognized (e.g., acute disseminated intravascular coagulation/'shock liver' with limb ischemia). HIT has features of both B-cell and T-cell immune responses; uptake of PF4/heparin complexes into macrophages ('macropinocytosis') facilitates the anti-PF4/heparin immune response. Antibody-induced activation of monocytes and platelets via their FcγIIA receptors triggers an intense procoagulant response. Sometimes, HIT antibodies recognize PF4 bound to (platelet-associated) chondroitin sulfate, explaining how HIT might occur without concurrent or recent heparin (delayed-onset HIT, 'spontaneous HIT syndrome'). The molecular structure of HIT antigen(s) has been characterized, providing a rationale for future drug design to avoid HIT and improve its treatment. The poor correlation between partial thromboplastin time and plasma argatroban levels (risking subtherapeutic anticoagulation) and need for intravenous administration of argatroban have led to increasing 'off-label' treatment with fondaparinux or one of the direct oral anticoagulants. Understanding the molecular mechanisms and unusual clinical features of HIT will improve its management.

Activated Protein C Resistance Does Not Increase Risk for Recurrent Stroke or Death in Stroke Patients.

BackgroundActivated protein C (APC) resistance is the most common inherited prothrombotic disorder. The role of APC resistance in ischemic stroke is controversially discussed.ObjectivesThe aim of this single center follow up study was to investigate the effect of APC resistance on stroke recurrence and survival in stroke patients.Patients/MethodsWe retrospectively identified 966 patients who had had an ischemic stroke or transitory ischemic attack (TIA) and in whom laboratory tests for APC resistance had been conducted. These patients were contacted to determine the primary outcomes of recurrent ischemic stroke or death.ResultsA total of 858 patients with an average follow up time of 8.48 years were included. APC resistance did not influence cumulative incidence functions for stroke free and total survival. In multivariate analyses, crude and adjusted hazard ratios for recurrent stroke as well as for death where not significantly increased in patients with APC resistance. This also applies to the subgroups of young patients, patients with cryptogenic stroke and patients with atrial fibrillation.ConclusionAPC-resistance is not a risk factor for subsequent stroke or death in patients with a first ischemic stroke or TIA. Testing for APC-resistance in stroke patients therefore cannot be routinely recommended.

A Targeted Photochemical Microfluidic Vascular Injury Model for in Vitro Thrombosis Studies: Usage in Heparin-Induced Thrombocytopenia (HIT)

Replicating the complexities of the human blood vessel include the establishment of a 3-D confluency of viable endothelial cells (ECs) on an appropriate matrix, use of human whole blood or specific components of blood, varied shear stresses, and the induction of a localized and controlled injury within the observable field to understand and intervene in hemostatic events. This array of complexities have made vascular modeling an important unmet challenge. Such a model would enhance our understanding of the pathogenesis of diverse coagulation disorders, such as the prothrombotic disorder HIT. In HIT, the platelet-rich clots lead to its other designation as the "white clot syndrome". To provide an improved injury model, we adapted a hematoporphyrin-photochemical injury using a Fluxion Bioflux microfluidic system. When illuminated with 405 nm light, hematoporphyrin releases reactive oxygen species, inducing localized EC injury within the exposed field, but without denuding ECs. Unlike rose bengal, hematoporphyrin does not cause fluorescent interference with quantitative analysis of the developing thrombus. We propose that the model permits refined analysis of ECs transitioning from areas of quiescence to injury to quiescence, allowing us to localize and quantify the contribution of various components to the growing thrombus. In HIT, patients form antibodies to complexes of the platelet-specific chemokine, platelet factor 4 (PF4), and negatively-charged molecules such as infused heparin and heparans found on the surface of platelets and monocytes. ECs also bind PF4 immune complexes due to their highly negatively charged glycocalyx-rich surface. Prior murine cremaster laser injury studies showed that HIT antibodies bind predominantly to the EC surface rather than platelet within the thrombus itself. Was this observation related to the nature of the cremaster injury? Would antibody binding to the EC lining also be important in a wholly human HIT detection system? Using the described photochemical microfluidic system, we created a localized injury in human umbilical vein EC-lined channels through which we flowed whole human blood. Activated platelets established growing aggregates at the site of EC injury, releasing more PF4 that then bound to non-activated ECs downstream of the injury. Whole blood containing a HIT-like antibody to simulate the prothrombotic state of HIT was then flowed over this injured vasculature. HIT antibodies and then platelets bound sequentially to this new site of HIT antigen, allowing the thrombus to propagate downstream. We have named this phenomenon "rolling barrage" and suggest that a key part of the prothrombotic nature of HIT lies in antibody-mediated activation of downstream EC with subsequent thrombus propagation. HIT often occurs in the setting in surgery, which might prime EC dysfunction. We therefore treated the ECs (TNF-α) prior to injury and introducing the HIT-like antibody. TNF-α activated the EC lining, leading to loss of the anticoagulant EC surface, which enhanced clot formation downstream of the site of photochemical injury. Therefore, in a prothrombotic state such as HIT, we propose that a local injury acts as a nidus for thrombus initiation. The procoagulant process spreads distally in part because of released PF4 adhering to the downstream EC glycocalyx, which is exacerbated by mediators of inflammation. We anticipate that the described model can be used to study novel interventions to block this cycle in a wholly human system with control over the contribution of individual cellular elements, and will further understanding of the importance of this mechanism in other prothrombotic disorders. DisclosuresNo relevant conflicts of interest to declare.