Sustained release of tissue factor following thrombosis of lower limb trauma.

This study was undertaken to provide evidence for the mechanism of venous thromboembolism (VTE) in healthy patients with minor lower limb injury (fracture; Achilles tendon rupture) that was medically managed with plaster cast/brace immobilization. The Plaster Cast clinical trial provided a unique opportunity to identify the natural history of VTE using placebo-controlled patients (n = 183) with validation of the mechanism using the low-molecular-weight heparin (LMWH; reviparin)-treated patients (n = 182). Confirmed VTE in this population was associated with a burst of tissue factor release (and a minor fibrinolytic deficit) leading to thrombin generation that was sustained at least 5 weeks, greater with fractures than with soft-tissue injuries and greater with surgery than with conservative treatment. The root cause likely involves platelet/leukocyte activation (inflammation) rather than endothelial cell injury. Thromboprophylaxis with a low dose of LMWH reduced thrombin generation, with patients undergoing surgery benefitting the most.

Emphasis on the Role of PF4 in the Incidence, Pathophysiology and Treatment of Heparin Induced Thrombocytopenia.

Heparin Induced Thrombocytopenia (HIT) is caused by antibodies that recognize platelet factor 4 (PF4) associated with polyanionic glycosaminoglycan drugs or displayed on vascular cell membranes. These antibodies are elicited by multimolecular complexes that can occur when heparin is administered in clinical settings associated with abundant PF4. Heparin binding alters native PF4 and elicits immune recognition and response. While the presence of heparin is integral to immunogenesis, the HIT antibody binding site is within PF4. Thus HIT antibodies develop and function to cause thrombocytopenia and/or thrombosis only in the presence of PF4. Future emphasis on understanding the biology, turnover and regulation of PF4 may lead to insights into the prevention and treatment of HIT.

A colorimetric, metabolic dye reduction assay detects highly activated platelets: application in the diagnosis of heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is caused by antibodies, elicited in response to heparin anticoagulant therapy, which can cause extreme platelet activation and result in a highly procoagulant state. Most diagnostic tests for HIT antibodies measure in vitro platelet activation by detecting aggregation or granule release responses. This study demonstrates that the high level of activation by HIT antibodies leads to a rapid breakdown of platelet metabolic activity. Resting or mildly activated platelets metabolize tetrazolium-based indicator dye to a dark colored product, in proportion to cell concentration and dye incubation time. Highly activated, procoagulant platelets resulting from incubation with HIT antibodies fail to metabolize dye and remain light in color. The loss of ability to metabolically reduce indicator dye provides a colorimetric endpoint that can be used in an in vitro washed platelet activation assay to detect HIT antibodies. In a prospective evaluation, 145 diagnostic specimens were tested concurrently by both the colorimetric, dye reduction assay and the clinical laboratory standard, radiolabeled-serotonin release assay ((14)C-SRA). Results were in agreement for 96-100% of cases, depending on the chosen stringency of assay cut-off values. This study demonstrates that the metabolic dye reduction assay is comparable to the (14)C-SRA for HIT diagnosis. In addition, this novel assay may have even wider applicability, facilitating studies on the physiologic and clinical relevance of highly activated platelet populations.

Physiological changes in membrane-expressed platelet factor 4: implications in heparin-induced thrombocytopenia.

BACKGROUND: Many heparin-induced thrombocytopenia (HIT) antibodies cause platelet activation in the serotonin release assay (SRA) in the absence of heparin. This in vitro observation may help unravel the mechanism of delayed-onset HIT, where seropositive patients develop thrombocytopenia and associated thrombosis after cessation of heparin. OBJECTIVE: Studies were conducted to examine the relationship between platelet environment, surface PF4 expression, and the extent of heparin-independent platelet activation in the SRA. METHODS: Ex vivo platelets were washed and labeled for SRA, then used either before or after 45 minutes of recovery at 37 degrees C. HIT antibody-mediated serotonin release in the absence of heparin was compared to the extent of surface staining of the platelets with fluorescent anti-human PF4 antibodies. RESULTS: Handling of platelets for in vitro studies resulted in transient expression of surface PF4, and it was during this interval that platelets were most sensitive to activation by HIT antibodies in the absence of heparin. Heparin-independent platelet activation was attenuated when SRA-positive specimens were retested after platelets were incubated 45 minutes at 37 degrees C. Surface PF4 expression was diminished on the rested platelets, compared to the same platelets labeled immediately after handling. Thus compared to rested platelets, mildly activated platelets had elevated surface PF4 expression and a higher level of HIT antibody-mediated, heparin-independent platelet activation. CONCLUSION: Surface expression of PF4 reflects HIT antigen presentation, and varies with the physiological state of platelets. Thus there can be differences in HIT antibody target availability among patients which may explain the variability in consequences of HIT antibody seropositivity.

Incidence of antiplatelet factor 4/heparin antibody induction in patients undergoing percutaneous coronary revascularization.

The incidence of antiplatelet factor-4/heparin antibody formation in patients who receive contemporary doses of unfractionated heparin in the setting of percutaneous coronary revascularization is unknown. Also unknown is the ability of these antibodies to activate platelets or adversely affect clinical outcome in the absence of clinically recognized heparin-induced thrombocytopenia. To address these questions, we serially measured antiplatelet factor-4/heparin antibody levels and performed serotonin release assays in patients who underwent percutaneous coronary intervention. Correlations were then made across antibody induction, heparin exposure, and clinical outcome at 6 months.

Prevalance of heparin-dependent platelet-activating antibodies in preterm newborns after exposure to unfractionated heparin.

Heparin is frequently used in preterm infants to prolong the patency of intravascular catheters. The aim of this study was to evaluate the prevalence of heparin-dependent platelet-activating antibodies in newborns. A cross-section of all preterm newborn infants expected to require heparin to maintain patency of a central venous access line were enrolled. A blood sample was obtained soon after birth before heparin exposure to exclude the possibility of placental transfer of maternal heparin-dependent platelet-activating antibodies. A second sample was obtained at termination of heparin use (mean duration of heparin exposure was 23 +/- 13 days; range, 6-67). Paired samples, at birth and after heparin use, were available for 42 infants with a mean gestational age of 27.8 +/- 2.2 weeks and birth weight of 1036 +/- 267 g. Thrombocytopenia developed in 57% (24/42) of the infants. None of these infants had clinical suspicion of thrombosis during the study period. The etiology of thrombocytopenia was confirmed sepsis in six, presumed sepsis in three, necrotizing enterocolitis in one, and unclear in 14 infants. Anti-heparin/platelet factor 4 antibodies measured using the standard assays for heparin-induced thrombocytopenia (two commercially available enzyme-linked immunosorbent assay tests and the functional platelet serotonin release assay) were negative on all infants. Although it could be related to the poor ability of these infants to mount an immunologic response, further research is necessary to fully understand this lack of response to heparin and to elucidate further the reasons for thrombocytopenia in very-low-birth-weight infants.

Decreased prevalence of heparin-induced thrombocytopenia with low-molecular-weight heparin and related drugs.

Heparin-induced thrombocytopenia (HIT) Type II represents a disease spectrum associated with a high risk of thrombosis leading to limb loss and death. The pathophysiology of HIT is based on the development of antibodies to the heparin-platelet factor 4 (PF4) complex. Unfractionated heparin (UFH) is heterogeneous in molecular chain length and degree of sulfation accounting in part, for, the heterogeneity of HIT antibodies. Because of its smaller size, low-molecular-weight heparin (LMWH) does not interact with PF4 and platelets as efficiently as does UFH. This translates into a lower risk of immune sensitization with LMWH than with UFH treatment. LMWH is less likely than UFH to cause antibody generation and thus patients do not develop clinical HIT at the same frequency with LMWH as with UFH treatment. The antibodies generated by LMWH treatment are more often immunoglobulin A (IgA) and IgM as opposed to IgG antibodies, which are associated with symptomatic clinical HIT generated by exposure to UFH. However, platelet activation/aggregation can occur from LMWHs in the presence of most pre-existing HIT antibodies that had been generated from UFH exposure, although the response is less than that caused by UFH plus HIT antibody. With the expanded use of LMWH, the frequency of clinical HIT may naturally decline, given that LMWHs are less likely to generate HIT antibody.

Newer insights on the mechanism of heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) type II is a complex clinical syndrome. It is an immune reaction to heparin in which the formation of antibodies targeted against the heparin-platelet factor 4 complex results in platelet activation. Platelet activation plays a central role in HIT; however, platelet activation does not occur as an isolated physiologic response. To elucidate further the mechanism of thrombogenesis in HIT, we undertook studies to determine the effect of heparin antibodies on endothelial cells, leukocytes, and the inflammatory state. We summarize our previous and new findings. For endothelial cells: Antiheparin antibodies bind to and directly activate microvascular endothelial cells, whereas binding to and activating macrovascular endothelial cells requires preactivation by platelets or tumor necrosis factor alpha (TNFalpha). Increased circulating levels of hemostatic activation factors as observed with thrombosis, particularly soluble P-selectin, plasminogen activator inhibitor type 1 (PAI-1), tissue factor, and thrombomodulin, were associated with endothelial cell activation and were also found in the blood circulation of patients with HIT. For the inflammatory state: Neutrophils and monocytes (but not lymphocytes) bind to and form complexes with platelets in the presence of HIT antibodies. Activated monocytes bind to endothelial cells and produce a procoagulant state. Patients with HIT have an increased level of cytokines in their blood circulation. For HIT antibodies: Only heparin fractions larger than 5 kd interacted with HIT antibodies, explaining why low-molecular-weight heparin (LMWH) usually does not generate antibodies. HIT antibodies are heterogeneous in structure, affinity, and specificity. These data suggest that, in addition to the platelet component, several other mechanisms are associated with the pathophysiology of HIT. These include an inflammatory state, endothelial cell remodeling, and the known procoagulant state. Differences between patients in the levels of the inflammatory markers may relate to various stages of the inflammatory/procoagulant state that exists in patients with HIT. The variations within the HIT antibodies may influence their ability to activate platelets, endothelial cells, and leukocytes, and thus contribute further to the variations in the pathogenicity of HIT.

Heparin-induced thrombocytopenia with associated thrombosis in children after the Fontan operation: report of two cases.

Heparin-induced thrombocytopenia is a widely recognized clinical disorder. The spectrum of disease ranges from clinically insignificant to severe thrombosis (heparin-induced thrombocytopenia with associated thrombosis). Overall, thrombosis occurs in approximately 33% of adults diagnosed with heparin-induced thrombocytopenia and has been associated with high morbidity and mortality rates. Diagnostic testing for this disorder is not standard in children with thrombocytopenia who are receiving heparin, despite the fact that children with congenital heart disease may be exposed to heparin frequently. There are few reported cases of heparin-induced thrombocytopenia with associated thrombosis in children; herein, we describe the cases of 2 children who developed this disorder after undergoing a Fontan operation.