Predicting Pulmonary Embolism in Total Joint Arthroplasty Patients A Pilot Study.

Postoperative venous thromboembolism (VTE) is a common and costly complication following total joint arthroplasty (TJA). Development of a refined thrombophilic screening panel will better equip clinicians to identify patients at high-est risk for developing VTEs. In this pilot study, 62 high-risk TJA recipients who had developed pulmonary emboli (PE) within 90-days of surgery were eligible to participate. Of these patients, 14 were enrolled and subsequently adminis-tered a pre-determined panel of 18 hematologic tests with the aim of identifying markers that are consistently elevated or deficient in patients developing PE. A separate cohort of seven high-risk TJA recipients who did not report a symp-tomatic VTE within 90-days of surgery were then enrolled and Factor VIII and lipoprotein(a) levels were assessed. The most common aberrance was noted in 10 patients (71.4%) who had elevated levels of Factor VIII followed by five patients (35.7%) who had elevated levels of lipoprotein(a). Factor VIII was significantly prevalent (p < 0.001) while lipoprotein(a) failed to achieve statistical significance (p = 0.0708). Of the patients who were within normal limits of Factor VIII, three-fourths were "high-normal" with Fac-tor VIII levels within 5% of the upper limit of normal. This study demonstrates the potential utility of this hematologic panel as part of a perioperative screening protocol aimed at identifying patients at risk for developing VTEs. However, future larger scale studies assessing the capabilities and limitations of our findings are warranted.

Reproducibility over time and effect of low-dose aspirin on soluble P-selectin and soluble CD40 ligand.

Platelet markers [soluble CD40 ligand (sCD40L) and soluble p selectin (sPselectin)] are associated with platelet activation and cardiovascular events. We sought to investigate the reproducibility of these markers over time and the effect of low-dose aspirin on sCD40L and sPselectin in plasma and serum. Following an overnight fast, 40 healthy volunteers had weekly phlebotomy and were administered aspirin 81 mg/day between weeks 3 and 4. Reproducibility over time was assessed by coefficient of variation (CV) and inter-class correlation coefficient. Correlation between markers was assessed using Pearson r statistic. Difference between levels pre- and post-aspirin was measured with Wilcoxon signed-rank test. Data are presented as median (interquartile range). sCD40L and sPselectin measurements were reproducible over time in plasma and serum (CV < 10 %). Measurement of sCD40L and sPselectin in plasma correlated with levels in serum before aspirin and after aspirin. There was no significant correlation between sCD40L and sPselectin. After 1-week of aspirin 81 mg/day, there was a reduction in sCD40L and sPselectin in serum and plasma, respectively. Soluble CD40L and sPselectin are independent markers that are reproducible over time in both plasma and sera and are reduced by 1-week of low-dose aspirin.

Platelet aggregation and coagulation factors in orthopedic surgery.

Hemostasis is a major concern during the perioperative period. Changes in platelet aggregation and coagulation factors may contribute to the delicate balance between thrombosis and bleeding. We sought to better understand perioperative hemostasis by investigating the changes in platelet aggregation and coagulation factors during the perioperative period. We performed a prospective cohort analysis of 70 subjects undergoing non-emergent orthopedic surgery of the knee (n = 28), hip (n = 35), or spine (n = 7) between August 2011 and November 2011. Plasma was collected preoperatively (T1), 1-h intraoperatively (T2), 1-h (T3), 24-h (T4) and 48-h (T5) postoperatively. Platelet function testing was performed using whole blood impedance aggregometry. Coagulation assays were performed for factor VII, factor VIII, von Willebrand Factor (vWF), and fibrinogen. Of the 70 patients, mean age was 64.1 ± 9.8 years, 61% were female, and 74% were Caucasian. Platelet activity decreased until 1 h postoperatively and then significantly increased above baseline at 24- and 48-h postoperatively. Compared to baseline, coagulation factors decreased intraoperatively. Factor VII activity continued to decrease, while FVIII, vWF, and fibrinogen all increased above baseline postoperatively. The results of our study indicate significant changes in platelet activity and coagulation factors during the perioperative period. Both platelet activity and markers of coagulation decrease during the intraoperative period and then some increase postoperatively. These changes may contribute to the hypercoagulabity and/or bleeding risk that occurs in the perioperative period. Future prospective studies aimed at correlating hemostatic changes with perioperative outcomes are warranted.

Aspirin attenuates platelet activation and immune activation in HIV-1-infected subjects on antiretroviral therapy: a pilot study.

BACKGROUND: Mechanisms for increased cardiovascular risk in HIV-1-infected adults are incompletely understood, but platelet activation and immune activation leading to a prothrombotic state have been proposed as significant contributors. Aspirin has antiplatelet and immunomodulatory properties. We explored whether 1 week of low-dose aspirin attenuates platelet activation and immune activation in HIV-1-infected and virologically suppressed adults on antiretroviral therapy. METHODS: Platelet activation and immune activation were measured in HIV-1-infected subjects virologically suppressed on antiretroviral therapy and controls before and after 1 week of low-dose aspirin. RESULTS: Compared with control subjects, HIV-1-infected subjects had increased platelet activation, as measured by spontaneous platelet aggregation and aggregation in response to adenosine diphosphate, collagen, and arachidonic acid. After aspirin therapy, percent aggregation decreased similarly in both HIV-1-infected and control subjects to all platelet agonists tested except aggregation in response to arachidonic acid, which remained elevated in the HIV-1-infected group. HIV-1-infected subjects exhibited increased markers of T-cell activation (CD38 and HLA-DR) and monocyte activation (sCD14), which decreased after 1 week of aspirin therapy. Moreover, leukocyte responses to Toll-like receptor stimulation were enhanced after 1 week of aspirin therapy. In vitro studies showed that HIV-1 plasma could activate healthy platelets, which in turn activated monocytes, implicating a direct role for activated platelets in immune activation. CONCLUSIONS: Our data demonstrate that heightened platelet activation and immune activation in treated HIV-1 disease are attenuated by 1 week of aspirin therapy. Aspirin should be further studied for its antithrombotic and immunomodulatory benefits in treated HIV-1 disease.

The inhibition effect of anti-GPIIIa49-66 antibody on megakaryocyte differentiation.

We previously reported that patients with early-onset HIV-1 ITP developed a unique anti-platelet integrin GPIIIa antibody against the GPIIIa49-66 epitope. Anti-GPIIIa49-66 antibody-induced platelet fragmentation requires sequential activation of the platelet 12-lipoxygenase (12-LO) and NADPH oxidase to release reactive oxygen species (ROS). 12-LO is upstream of the NADPH oxidase pathway and 12(S)-HETE, the product of 12-LO, induces the same oxidative platelet fragmentation as anti-GPIIIa49-66. Since the megakaryocyte (MK) is the progenitor cell for platelets, we have investigated the effect of anti-GPIIIa49-66 on MK differentiation and, in particular, the potential role of anti-GPIIIa49-66 induced ROS in this process. We first show that polyclonal anti-GPIIIa49-66 antibody isolated from HIV-1 ITP patients inhibits MK proliferation 2.5-fold in in vitro culture of human cord blood CD34+ cells driven by thrombopoietin (TPO). We also observe a three-fold decrease in the number of MK colony-forming units in the presence of a human monoclonal anti-GPIIIa49-66 antibody. However, we could not detect ROS release in DCFH-loaded mouse megakaryoblastic cells L8057 treated with anti-GPIIIa49-66 antibody. In addition, 12(S)-HETE does not inhibit the in vitro differentiation of L8057 cells induced by TPO. In fact, we found a dose dependent increase in the percentage of CD41 positive cells (from 17.1% to 48.7%) in in vitro culture of L8057 cells treated with various concentrations of H2O2 (from 5 to 20 µM). We therefore conclude that the anti-GPIIIa49-66 antibody inhibits MK differentiation through ß3 integrin signalling independent of ROS release.

C-terminal ADAMTS-18 fragment induces oxidative platelet fragmentation, dissolves platelet aggregates, and protects against carotid artery occlusion and cerebral stroke.

Anti-platelet integrin GPIIIa49-66 antibody (Ab) induces complement-independent platelet oxidative fragmentation and death by generation of platelet peroxide following NADPH oxidase activation. A C-terminal 385-amino acid fragment of ADAMTS-18 (a disintegrin metalloproteinase with thrombospondin motifs produced in endothelial cells) induces oxidative platelet fragmentation in an identical kinetic fashion as anti-GPIIIa49-66 Ab. Endothelial cell ADAMTS-18 secretion is enhanced by thrombin and activated by thrombin cleavage to fragment platelets. Platelet aggregates produced ex vivo with ADP or collagen and fibrinogen are destroyed by the C-terminal ADAMTS-18 fragment. Anti-ADAMTS-18 Ab shortens the tail vein bleeding time. The C-terminal fragment protects against FeCI3-induced carotid artery thrombosis as well as cerebral infarction in a postischemic stroke model. Thus, a new mechanism is proposed for platelet thrombus clearance, via platelet oxidative fragmentation induced by thrombin cleavage of ADAMTS-18.

Role of molecular mimicry of hepatitis C virus protein with platelet GPIIIa in hepatitis C-related immunologic thrombocytopenia.

Patients with HIV-1 immune-related thrombocytopenia (HIV-1-ITP) have a unique Ab against platelet GPIIIa49-66 capable of inducing oxidative platelet fragmentation in the absence of complement. HIV-1-seropositive drug abusers are more prone to develop immune thrombocytopenia than non-drug abusers and have a higher coinfection with hepatitis C virus (HCV) than non-drug abusers (90% vs 30%). Molecular mimicry was sought by screening a phage peptide library with anti-GPIIIa49-66 antibody as bait for peptides sharing homology sequences with HCV. Several phage peptide clones had 70% homology with HCV protein. Sera from dually infected thrombocytopenic patients with HCV and HIV-ITP reacted strongly with 4 nonconserved peptides from HCV core envelope 1. Reactivity correlated inversely with platelet count (r(2) = 0.7, P < .01). Ab raised against peptide PHC09 in GPIIIa(-/-) mice induced thrombocytopenia in wild-type mice. Affinity-purified IgG against PHC09 induced oxidative platelet fragmentation in vitro. Drug abusers dually infected with HCV and HIV-1 had a greater incidence and severity of thrombocytopenia as well as titer of anti-GPIIIa49-66/PHC09 Ab. NZB/W F1 mice injected with recombinant core envelope 1 developed Ab versus PHC09 and significantly decreased their platelet count (P < .001). Thus, HCV core envelope 1 can induce thrombocytopenia by molecular mimicry with GPIIIa49-66.

Platelet particle formation by anti GPIIIa49-66 Ab, Ca2+ ionophore A23187, and phorbol myristate acetate is induced by reactive oxygen species and inhibited by dexamethasone blockade of platelet phospholipase A2, 12-lipoxygenase, and NADPH oxidase.

An HIV antibody (Ab) against platelet integrin GPIIIa49-66 induces complement-independent platelet particle formation by the elaboration of reactive oxygen species (ROS) downstream of the activation of the platelet NADPH oxidase by the 12-lipoxygenase (12-LO) product 12(S)-HETE. To determine whether other inducers of platelet particle formation also function via the induction of ROS, we examined the effects of the Ca(2+) ionophore A23187 and phorbol myristate acetate (PMA). Both agents induced oxidative platelet particle formation in an identical fashion as Ab, requiring Ca(2+) flux and 12(S)-HETE production as well as intact NADPH oxidase and 12-LO pathways. Since HIV-ITP patients with this Ab correct their platelet counts with dexamethasone (Dex), we examined the role of this steroid in this unique autoimmune disorder. Dex at therapeutic concentrations inhibited Ab-, A23187-, or PMA-induced platelet particle formation by inhibiting platelet PLA(2), 12-LO, and NADPH oxidase. The operational requirement of translocation of PLA(2), 12-LO, and NADPH oxidase components (p67 phox) from cytosol to membrane for induction of ROS was both inhibited and partially reversed by Dex in platelets. We conclude that (1) platelet particle formation can be induced by the generation of ROS; and (2) platelet PLA(2), 12-LO, NADPH oxidase, and cytosol membrane translocation, requirements for ROS production, are inhibited by Dex.