Gowthami M. Arepally

Platelet functions beyond hemostasis

Summary.  Although their central role is in the prevention of bleeding, platelets probably contribute to diverse processes that extend beyond hemostasis and thrombosis. For example, platelets can recruit leukocytes and progenitor cells to sites of vascular injury and inflammation; they release proinflammatory and anti‐inflammatory and angiogenic factors and microparticles into the circulation; and they spur thrombin generation. Data from animal models suggest that these functions may contribute to atherosclerosis, sepsis, hepatitis, vascular restenosis, acute lung injury, and transplant rejection. This article represents an integrated summary of presentations given at the Fourth Annual Platelet Colloquium in January 2009. The process of and factors mediating platelet–platelet and platelet–leukocyte interactions in inflammatory and immune responses are discussed, with the roles of P‐selectin, chemokines and Src family kinases being highlighted. Also discussed are specific disorders characterized by local or systemic platelet activation, including coronary artery restenosis after percutaneous intervention, alloantibody‐mediated transplant rejection, wound healing, and heparin‐induced thrombocytopenia.

Prevalence of Heparin-Associated Antibodies Without Thrombosis in Patients Undergoing Cardiopulmonary Bypass Surgery

BACKGROUND Patients with cardiovascular disease almost invariably receive heparin before cardiopulmonary bypass surgery, which places them at risk of developing heparin-associated antibodies with a risk of thromboembolic complications. This study was designed to determine the prevalence of heparin-induced antibodies in patients before and after cardiopulmonary bypass surgery. METHODS AND RESULTS Plasma from 111 patients was tested before surgery and 5 days after surgery for heparin-dependent platelet-reactive antibodies with a 14C-serotonin-release assay (SRA) and for antibodies to heparin/platelet factor 4 complexes with an ELISA. Heparin exposure after surgery was minimized. Heparin-dependent antibodies were detected before surgery in 5% of patients with SRA and 19% of patients with ELISA. By the fifth postoperative day, there was a marked increase in patients positive on the SRA or ELISA (13% and 51%, respectively; P < .01 for each). Patients who had received heparin therapy earlier in their hospitalization were more likely to have a positive ELISA before surgery (35%; P = .017) and a positive ELISA (68%; P = .054) or SRA (30%; P = .002) after surgery. However, there was no difference in the prevalence of thrombocytopenia or thromboembolic events between the antibody-positive and-negative groups. CONCLUSIONS Approximately one fifth of patients undergoing cardiopulmonary bypass surgery have heparin-induced platelet antibodies detectable before the procedure as a result of prior heparin exposure, and many more develop antibodies after surgery. The absence of an association between these antibodies and thromboembolic complications in this study may be, in part, attributable to careful avoidance of heparin after surgery. The high prevalence of heparin-induced antibodies in this setting suggests that these patients may be at risk of developing thrombotic complications with additional heparin exposure.

Acquired von Willebrand Syndrome in Continuous-Flow Ventricular Assist Device Recipients

BACKGROUND Bleeding is a major cause of morbidity in recipients of continuous-flow left ventricular assist devices (CF-LVAD). A better understanding of the impact of CF-LVAD support on the hemostatic profile is necessary to establish better strategies for anticoagulation therapy and risk assessment for bleeding complications. A prospective multicenter study was conducted to characterize von Willebrand factor (vWF) profiles in patients undergoing CF-LVAD implantation. METHODS Blood samples were collected before and after CF-LVAD implantation from 37 patients between July 2008 and April 2009 at Duke University and the University of Minnesota. Blood samples were analyzed for vWF, platelet and collagen-binding ability. The presence of high-molecular-weight (HMW) vWF multimers were detected through gel electrophoresis, and deficiency was graded on a scale of 0 (normal) to 3 (severe loss). RESULTS All 37 patients exhibited significant loss of HMW vWF multimers within 30 days of CF-LVAD implantation. Ten of the 37 patients experienced bleeding complications after CF-LVAD placement. CONCLUSIONS All CF-LVAD recipients had acquired von Willebrand syndrome after LVAD placement, demonstrated by reduced or absent HMW vWF multimer levels. However, not all recipients had bleeding complications. These findings suggest that loss of HMW vWF multimers alone cannot predict bleeding risk. Further refinement of laboratory techniques and a larger follow-up is required to identify risk factors for bleeding in CF-LVAD recipients.

The HIT Expert Probability (HEP) Score: a novel pre‐test probability model for heparin‐induced thrombocytopenia based on broad expert opinion

Summary.  Background: The diagnosis of heparin‐induced thrombocytopenia (HIT) is challenging. Over‐diagnosis and over‐treatment are common. Objectives: To develop a pre‐test clinical scoring model for HIT based on broad expert opinion that may be useful in guiding clinical decisions regarding therapy. Patients/methods: A pre‐test model, the HIT Expert Probability (HEP) Score, was constructed based on the opinions of 26 HIT experts. Fifty patients referred to a reference laboratory for HIT testing comprised the validation cohort. Two hematology trainees scored each patient using the HEP Score and a previously published clinical scoring system (4 T’s). A panel of three independent experts adjudicated the 50 patients and rendered a diagnosis of HIT likely or unlikely. All subjects underwent HIT laboratory testing with a polyspecific HIT ELISA and serotonin release assay (SRA). Results: The HEP Score exhibited significantly greater interobserver agreement [intraclass correlation coefficient: 0.88 (95% CI 0.80–0.93) vs. 0.71 (0.54–0.83)], correlation with the results of HIT laboratory testing and concordance with the diagnosis of the expert panel (area under receiver‐operating curve: 0.91 vs. 0.74, P = 0.017) than the 4 T’s. The model was 100% sensitive and 60% specific for determining the presence of HIT as defined by the expert panel and would have allowed for a 41% reduction in the number of patients receiving a direct thrombin inhibitor (DTI). Conclusion: The HEP Score is the first pre‐test clinical scoring model for HIT based on broad expert opinion, exhibited favorable operating characteristics and may permit clinicians to confidently reduce use of alternative anticoagulants. Prospective multicenter validation is warranted.

Comparative analysis of von Willebrand factor profiles in pulsatile and continuous left ventricular assist device recipients.

A higher rate of nonsurgical bleeding has been observed in nonpulsatile left ventricular assist device (LVAD) recipients. von Willebrand factor (vWF) profiles were compared for nonpulsatile and pulsatile LVAD recipients to explore mechanisms that may contribute to the development of postimplant nonsurgical bleeding. The nonpulsatile mechanism may impair vWF function by creating a deficiency in vWF high molecular weight multimers (HMWMs), essential for hemostasis. High molecular weight multimer deficiency should result in low ristocetin cofactor (RCo) to vWF antigen ratios (vWF:RCo/vWF:Ag) because of impaired platelet (plt)-binding ability. von Willebrand factor profiles and HMWM were measured pre- and post-LVAD placement in 11 nonpulsatile (HeartMate II [HM II[) and 3 pulsatile (HeartMate XVE [HM XVE]) recipients. All the nonpulsatile LVAD recipients exhibited loss of HMWM 30 days postimplant. The vWF:RCo/vWF:Ag ratio was significantly lower after LVAD placement in the nonpulsatile group when compared with the pulsatile group. In addition, the vWF:RCo/vWF:Ag ratio decreased significantly from baseline 30 days postimplant within the nonpulsatile recipients. All nonpulsatile LVAD recipients had low vWF:RCo/vWF:Ag ratios 30 days post-LVAD even if the values were normal at baseline. These data suggest that nonpulsatile HM II recipients develop HMWM loss and impaired vWF platelet (plt)-binding ability after LVAD placement. Similar results were not observed in our small series of pulsatile HM XVE recipients. This finding could suggest a contributing factor to the increase in nonsurgical bleeding observed in nonpulsatile LVAD patients. Further investigation is ongoing to identify specific causes of vWF impairment.

Tissue factor around dermal vessels has bound factor VII in the absence of injury

Summary.  Background: ‘Idling’ or ongoing low‐level activity of the tissue factor (TF) pathway is a postulated mechanism by which the coagulation process can become active without a lag period at sites of injury. Objective: To determine whether TF around cutaneous vessels has bound factor VIIa in the absence of injury, and thus could participate in the idling process. Methods: Immunostaining of mouse skin with antibodies against a 15‐residue peptide from the sequence of mouse TF, and against the whole extracellular portion of TF. Results: The whole TF antibody recognized TF in squamous epithelium and around vessels in the dermis. By contrast, the monospecific antibody only recognized TF in the squamous epithelium, but not around vessels. We also found that biotinylated, active site‐inhibited FVIIa (FVIIai) bound to tissue sections in the same areas in which TF was recognized by the monospecific antibody (squamous epithelium), but did not bind around vessels. Molecular modeling revealed that FVIIa and FX binding to TF masked a significant part of the surface of the target peptide. Conclusions: In the aggregate, these data are most consistent with the interpretation that TF in perivascular sites has bound FVIIa, even in the absence of any injury. The presence of endogenously bound FVIIa prevents the subsequent binding of the monospecific antibody or exogenous FVIIai to perivascular TF.

Heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an immune-mediated hypercoagulable disorder caused by antibodies to platelet factor 4 (PF4) and heparin. HIT develops in temporal association with heparin therapy and manifests either as an unexplained thrombocytopenia or thrombocytopenia complicated by thrombosis. The propensity for thrombosis distinguishes HIT from other common drug-induced thrombocytopenias. Diagnosing HIT in hospitalized patients is often challenging because of the frequency of heparin use, occurrence of thrombocytopenia from other causes, and development of asymptomatic PF4/heparin antibodies in patients treated with heparin. This review summarizes our current understanding of the pathogenesis, clinical features, diagnostic criteria, and management approaches in HIT.

Anticoagulation Techniques in Apheresis: From Heparin to Citrate and Beyond

Anticoagulation is essential for maintaining the fluidity of extravascular blood on the apheresis circuit. Although both citrate and heparin are used as an anticoagulant during apheresis, citrate is preferred for the majority of exchange procedures because of its safety and effectiveness. Complications of citrate are primarily due to physiologic effects of hypocalcemia. Symptoms of hypocalcemia and other citrate‐induced metabolic abnormalities affect neuromuscular and cardiac function and range in severity from mild dysesthesias (most common) to tetany, seizures, and cardiac arrhythmias. Oral or intravenous calcium supplementation is advised for decreased ionized calcium levels and/or symptomatic management of hypocalcemia. Heparin‐based anticoagulation is limited to certain apheresis procedures (membrane‐based plasma exchange, LDL apheresis, or photopheresis) or is used in combination with citrate to reduce citrate load. While effective, heparin anticoagulation is associated with an increased frequency of bleeding complications and heparin‐induced thrombocytopenia. J. Clin. Apheresis 2012.

Plasmapheresis and heparin reexposure as a management strategy for cardiac surgical patients with heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) complicates the management of patients presenting for cardiac surgery, because high-dose heparin anticoagulation for cardiopulmonary bypass is contraindicated in these patients. Alternative anticoagulants are available, but there are concerns about dosing, efficacy, monitoring, thrombosis, and hemorrhage. METHODS: A retrospective chart review between November 2004 and March 2008 retrieved perioperative clinical and laboratory data for 11 adult cardiac surgical patients with a preoperative history of HIT and a current positive antiheparin/platelet factor 4 (anti-HPF4) antibody titer, who were managed with plasmapheresis and heparin anticoagulation. RESULTS: The median (interquartile range) preoperative anti-HPF4 antibody titer was 0.8 (0.7–2.2). Three of the 11 patients (27%) died of causes unrelated to HIT and 1 of these patients (9%) developed an ischemic foot, in the setting of cardiogenic shock, not thought to be HIT-related. A single plasmapheresis treatment reduced titers by 50%–84%, and 6 patients had negative titers after treatment; none of the 3 patients with reduced titers developed clinical HIT. CONCLUSIONS: This case series describes an alternative management strategy using intraoperative plasmapheresis for patients presenting for cardiac surgery with acute or subacute HIT. Reducing antibody load can potentially decrease the thrombotic risk associated with high anti-HPF4 titers and decrease the urgency to initiate postoperative anticoagulation in this patient group at high risk of postoperative bleeding.

Dynamic antibody-binding properties in the pathogenesis of HIT

Rapid laboratory assessment of heparin-induced thrombocytopenia (HIT) is important for disease recognition and management. The utility of contemporary immunoassays to detect antiplatelet factor 4 (PF4)/heparin antibodies is hindered by detection of antibodies unassociated with disease. To begin to distinguish properties of pathogenic anti-PF4/heparin antibodies, we compared isotype-matched monoclonal antibodies that bind to different epitopes: KKO causes thrombocytopenia in an in vivo model of HIT, whereas RTO does not. KKO binding to PF4 and heparin is specifically inhibited by human HIT antibodies that activate platelets, whereas inhibition of RTO binding is not differentially affected. Heparin increased the avidity of KKO binding to PF4 without affecting RTO, but it did not increase total binding or binding to nontetrameric PF4(K50E). Single-molecule forced unbinding demonstrated KKO was 8-fold more reactive toward PF4 tetramers and formed stronger complexes than RTO, but not to PF4(K50E) dimers. KKO, but not RTO, promoted oligomerization of PF4 but not PF4(K50E). This study reveals differences in the properties of anti-PF4 antibodies that cause thrombocytopenia not revealed by ELISA that correlate with oligomerization of PF4 and sustained high-avidity interactions that may simulate transient antibody-antigen interactions in vivo. These differences suggest the potential importance of epitope specificity in the pathogenesis of HIT.