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J M Walenga

Publications and source records attributed to J M Walenga.

At least 19 recordsLinked to original sources
PubMed2005

Activation of platelets by heparin-induced thrombocytopenia antibodies in the serotonin release assay is not dependent on the presence of heparin.

The serotonin release assay (SRA) tests for antibodies responsible for heparin-induced thrombocytopenia (HIT). By definition, SRA-positive antibodies cause platelet serotonin release in vitro, in the presence of low concentrations of heparin, but not with excess heparin. Many SRA-positive sera activate platelets in the presence of saline without drug, either as a result of residual heparin in the specimen, or because of intrinsic features of the HIT antibodies. The present experiments show that neither exhaustive heparinase treatment, nor chromatographic removal of heparin abrogates the spontaneous platelet activation caused by these HIT antibodies. This is the first study to systematically demonstrate that in vitro activity of HIT antibodies can be independent of heparin. In addition, T-gel chromatography demonstrated differences among fractions of enzyme-linked-immunosorbent assay (ELISA)-positive HIT antibodies within individual specimens. Certain ELISA-positive fractions had SRA activity while others did not, and the SRA activity was not proportional to HIT antibody ELISA titer. These data suggest that antibodies formed as a result of heparin treatment are heterogeneous, and that some can contribute to the pathogenesis of HIT even when heparin is no longer present.

Antibodies↗
PubMed2001

Inhibition of tissue factor-activated platelets by low-molecular-weight heparins and glycoprotein IIb/IIIa receptor antagonist.

Thrombotic disorders can lead to vascular distress and platelet activation eventually resulting in the rupture of the lesions where a sizable amount of tissue factor (TF) is generated during the pathogenesis of arterial diseases. Since low-molecular-weight heparins (LMWHs) and platelet glycoprotein (GP) IIb/IIIa inhibitors are clinically used for the management of acute coronary syndrome (ACS), studies were taken to determine the effects of these agents on TF-mediated activation of platelets. Freshly drawn native whole blood (WB) from normal healthy volunteers (n = 6) supplemented with a predetermined amount of TF was incubated with equivalent anti-Xa adjusted amounts of various LMWHs at 0.01-1.0 U/ml and tirofiban from 10 to 100 ng/ml. Platelet activation was assessed by measuring the expression of P-selectin (CD62) and the generation of platelet aggregates. At 0.01 U/ml, enoxaparin exhibited a stronger inhibition of TF-induced platelet activation compared to ardeparin and dalteparin. At 0.1 U/ml, these LMWHs produced a comparable inhibition of total P-selectin expression, and at 1.0 U/ml, a marked inhibition was noted. Since enoxaparin produced the best concentration-dependent inhibition of P-selectin expression (saline: 76 +/- 10% vs. 1.0 U/ml enoxaparin: 18 +/- 7%; P < .02) and platelet aggregate formation (saline: 63 +/- 7% vs. 1.0 U/ml enoxaparin: 35 +/- 6%, P < .035), this agent was used for additional studies. Unlike enoxaparin, tirofiban produced a weak concentration-dependent inhibition of platelet activation. At 100 ng/ml, tirofiban produced a 40% inhibition of P-selectin expression and about 60% inhibition of platelet aggregate formation. To elucidate the potential interaction between tirofiban and enoxaparin, the effect of 10 and 100 ng/ml tirofiban was studied with enoxaparin-supplemented WB in a 0.01-1.0 U/ml range. Additive effects between these two agents were noted only at lower concentrations. Thus, at therapeutic concentrations (0.8-1.2 U/ml), enoxaparin itself was capable of inhibiting TF-mediated activation of platelets to > 70%; whereas tirofiban failed to produce such concentration-dependent inhibition. This suggests that the simultaneous administration of GPIIb/IIIa receptor antagonist with LMWH may not have any added benefit in the clinical management of patients with ACS.

Blood Platelets↗
PubMed2001

Argatroban anticoagulant therapy in patients with heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is an immune-mediated syndrome caused by heparin. Complications range from thrombocytopenia to thrombocytopenia with thrombosis. We report a prospective, historical- controlled study evaluating the efficacy and safety of argatroban, a direct thrombin inhibitor, as anticoagulant therapy in patients with HIT or HIT with thrombosis syndrome (HITTS). METHODS AND RESULTS: Patients with HIT (isolated thrombocytopenia, n=160) or HITTS (n=144) received 2 microgram. kg(-1). min(-1) IV argatroban, adjusted to maintain the activated partial thromboplastin time 1.5 to 3.0 times baseline value. Treatment was maintained for 6 days, on average. Clinical outcomes over 37 days were compared with those of 193 historical control subjects with HIT (n=147) or HITTS (n=46). The incidence of the primary efficacy end point, a composite of all-cause death, all-cause amputation, or new thrombosis, was reduced significantly in argatroban-treated patients versus control subjects with HIT (25.6% versus 38.8%, P=0.014). In HITTS, the composite incidence in argatroban-treated patients was 43.8% versus 56.5% in control subjects (P=0.13). Significant between-group differences by time-to-event analysis of the composite end point favored argatroban treatment in HIT (P=0.010) and HITTS (P=0.014). Argatroban therapy, relative to control subjects, also significantly reduced new thrombosis and death caused by thrombosis (P<0.05). Argatroban-treated patients achieved therapeutic activated partial thromboplastin times generally within 4 to 5 hours of starting therapy and, compared with control subjects, had a significantly more rapid rise in platelet counts (P=0.0001). Bleeding events were similar between groups. CONCLUSIONS: Argatroban anticoagulation, compared with historical control subjects, improves clinical outcomes in patients who have heparin-induced thrombocytopenia, without increasing bleeding risk.

Aged↗
PubMed2001

The impact of antithrombin (H) concentrate infusions on pulmonary function in the acute phase of thermal injury.

BACKGROUND: Pulmonary complications occur frequently after thermal injury. OBJECTIVE: This open pilot study was performed as an initial assessment of the safety and efficacy of antithrombin H [AT(H)] concentrate in ameliorating the respiratory morbidity during the acute phase of injury. MATERIALS & METHODS: Thirty-two patients were eligible for the study; of these, nine opted for treatment with q8 h [AT(H)]. The mean daily peak values of pulmonary parameters such as PaO(2)/FiO(2) ratio, and RAW scores were computed for days 1-8. RESULTS: Control and AT(H)-treated patients were similar in age, % total burn surface area, inhalation injury, and mortality. Forty-three percent of the burn controls, and 23% of the AT(H)-treated patients had pneumonia, p<0.01. The median hospital stay for both groups was 42 days; however, the median number of ventilatory days for burn controls was 23 days vs 10 days for AT(H)-treated patients. The AT(H)-treated patients had admission AT plasma levels of 46+/-14% vs 49+/-18% in burn controls, (normal=100+/-20%). The AT plasma level was maintained at 120+/-24% in the AT(H)-treated patients vs 50+/-15% in the burn control group for the first four days following the acute injury, p<0.002. Thrombate(R) concentrate infusions were, in general, well tolerated by patients. The median dose was 97 u/kg/dose q8 h. Compared to burn controls, AT(H)-treated patients had higher PaO(2)/FiO(2) ratios between days 4-6, p<0.01. In comparing these two groups with and without inhalation, airway resistance (assessed by the RAW score) was significantly lower in the AT(H)-treated group with inhalation compared to the burn controls with inhalation on days 2 and 6, p<0.02. CONCLUSIONS: With a trend toward decreased airway resistance during AT(H) concentrate infusions, and increased oxygenation, AT(H)-treated patients had significantly fewer episodes of pneumonia compared to controls. AT(H) concentrates may modify the impact of thrombin on acute inflammation, and improve respiratory function in the acute phase of thermal injury.

Adult↗
PubMed2001

The hemostatic effects of warfarin titration in post CABG patients in comparison to placebo treatment.

BACKGROUND: Since coronary artery bypass graft patients remain at risk of coronary artery and bypass graft occlusion after successful surgery, adjunct treatment regimens are under investigation. In a study of the patients of the multicenter Post Coronary Artery Bypass Graft (Post CABG) Trial, 1 mg warfarin was found to have no important effect on coagulation parameters. STUDY DESIGN: The effects of 1, 2 and 3 mg warfarin were evaluated at six-week intervals in 20 Post CABG Trial patients receiving titrated dose increases in comparison to 20 patients of similar age, gender and time from CABG treated with placebo. RESULTS: International normalized ratio (INR) values increased with warfarin dose increments for 1, 2, and 3 mg, respectively (0.95+/-0.16, 1.08+/-0.19, and 1.34+/-0.39) and in comparison to placebo treated patients (dosextreatment p<0.001). Factor VII coagulant activity decreased with warfarin titration (1 mg, 119.0+/-18.3 %; 2 mg, 100.6+/-32.8 %; 3 mg, 95.0+/-27.8 %) and in comparison to placebo (dosextreatment p=0.008). Levels of prothrombin fragment F1.2, tissue plasminogen activator, fibrinogen and von Willebrand factor were unchanged with warfarin dose increments and in comparison to placebo. CONCLUSIONS: At doses up to 3 mg, warfarin acts on the INR through a reduction of factor VII with no effect on the fibrinolytic system, fibrinogen or von Willebrand factor. At these doses F1.2 did not document reduced coagulation activity. The observations of this study were consistent with the decision in the Post CABG Trial to increase the warfarin dose above 1 mg to achieve a distinct effect of warfarin that was less than full anticoagulation.

Adult↗
PubMed2001

Effects of a heparin-binding protein on blood coagulation and platelet function.

The objective of this study was to characterize the heparin-binding properties of a protein secreted by mouse myeloma cells. The characterization was performed using clinical assays, such as heparin activity assays and heparin-induced thrombocytopenia (HIT) platelet activation assays. The tests were performed in the presence of heparin, low-molecular-weight heparins (LMWH), or heparinoids and either heparin-binding protein (HBP) or saline to determine whether the HBP affects the activity of heparins. The characterization of the HBP using heparin activity assays showed that the HBP shortened the prolonged clotting times of the activated partial thromboplastin time (aPTT) and thrombin clotting time induced by high concentrations of unfractionated heparin. The chromogenic assays for antithrombin (AT), thrombin inhibition, and factor Xa inhibition demonstrated that this effect is related to heparin concentrations below 0.5 IU/ml. The Heptest assay did not detect these differences. The HBP did not modify the anticoagulant effect of any LMWH or low- or high-sulfated glycosaminoglycans in the aPTT assay. Activation of donor platelets in the presence of unfractionated heparin, platelet factor 4 (PF4), and HIT-serum was not counteracted by the HBP in any of the assays. The characterization of the HBP using a PF4-enzyme-linked immunosorbent assay (ELISA) confirmed the lack of structural identity with PF4. However, the optical density data indicated that the protein structure may be similar to PF4 by binding to a PF4 antibody. These data suggest that the HBP isolated from mouse myeloma cells has a low affinity to heparin and interacts with the secondary binding site to AT and also perhaps to PF4.

Animals↗
PubMed2001

Evaluation of a new point of care heparin test for cardiopulmonary bypass: the TAS heparin management test.

Patients undergoing cardiopulmonary bypass (CPB) require anticoagulation with heparin to avoid thrombosis within the bypass circuit. The common method used to monitor the degree of anticoagulation is the activated clotting time (ACT). We evaluated a novel point of care device, the TAS (Pharmanetics, Raleigh, NC, USA) heparin management test (HMT), for its suitability in monitoring anticoagulation during CPB. In vitro analysis showed a dose-response (r2=0.988) of the HMT from 0.078-10.0 U/ml heparin, covering the range of heparin used during cardiac surgery (2-5 U/ml). Fifty randomly selected patients undergoing CPB were studied. Preheparin clotting times for these patients were 143+/-32 s for the HMT and 146+/-18 s for the ACT; 435+/-60 s HMT and 438+/-39 s ACT during CPB; 145+/-50 s HMT and 128+/-14 s ACT post-protamine (r2=0.797). epsilon-Aminocaproic acid treatment for inhibition of fibrinolysis did not affect the HMT. We conclude that the HMT correlates well with the ACT and may be useful for monitoring heparin during CPB. Advantages of the HMT are small sample volume and good sensitivity to heparin.

Anticoagulants↗
PubMed2001

Synthetic heparin pentasaccharide depolymerization by heparinase I: molecular and biological implications.

A synthetic pentasaccharide (SR90107/ ORG31540) representing the antithrombin III (ATIII) binding sequence in heparin is under clinical development for the prophylaxis and management of venous thromboembolism. This pentasaccharide exhibits potent anti-factor Xa (AXa) effects (>750 IU/mg) and does not exhibit any anti-factor IIa (AIIa) activity. Previous reports have suggested that synthetic heparin pentasaccharides are resistant to the digestive effects of heparinase I. To investigate the effect of heparinase I on the AXa activity of pentasaccharide SR90107/ORG31540, graded concentrations (1.25-100 microg/ml) were incubated with a fixed amount of heparinase I (0.1 U/ml). Heparinase I produced a strong neutralizing effect on this pentasaccharide, as measured by AXa activity. This observation led to further studies where high performance liquid chromatography (HPLC) analysis was employed to determine the potential breakdown products of the pentasaccharide. The experiment with the pentasaccharide included incubation (37 degrees C) at 1 mg/ml and exposure to graded concentrations of heparinase I (0.125-1 U/ml). After 30 min of incubation, the enzymatic activity was stopped by heat treatment and the mixture was analyzed using high performance size exclusion chromatography (HPSEC). Heparinase I concentration-dependent cleavage of the pentasaccharide was evident. The breakdown products exhibited a mass of 1,034 d and 743 d, respectively, suggesting the generation of a trisaccharide and a disaccharide moiety. The extinction of a disaccharide moiety in the UV region was high, indicating the presence of a double bond in this molecule. These data clearly suggest that pentasaccharide SR90107/ORG31540 is digestible by heparinase I into its two components. Furthermore, these data support the hypothesis that heparinase I can be used as a neutralizing agent for pentasaccharide overdose. Additionally, a highly methylated analog of the previously mentioned synthetic pentasaccharide. SanOrg34006, which has also been subjected to similar experiments, has shown complete resistance to the depolymerizing function of heparinase I; therefore, its use may be appropriate in chronic situations as a long-acting form of the pentasaccharide.

Antithrombin III↗
PubMed2001

Emerging anticoagulant and thrombolytic drugs.

Since its discovery, heparin has been used intensely as an anticoagulant for several medical and surgical indications. However, efforts are in progress to replace heparin because of its serious complications, such as intraoperative and postoperative bleeding, osteoporosis, alopecia, heparin resistance, heparin rebound, heparin-induced thrombocytopenia (HIT) and thrombosis syndrome (HITTS), and other disadvantages. Significant developments in the field of new anticoagulants have resulted in the evaluation and introduction of low molecular weight heparins (LMWHs) and heparinoids, hirudin, ancrod, synthetic peptides and peptidomimetics. However, despite significant progress in the development of these new anticoagulants, a better or an ideal anticoagulant for cardiovascular patients is not yet available and heparin still continues to amaze both basic scientists and the clinicians. To minimise the adverse effects of heparin, newer approaches to optimise its use in combination with the new anticoagulants may provide better clinical outcome. In our experience, the off-label use of argatroban at a dose of 300 microg/kg iv. bolus followed by 10 microg/kg/minute infusion in combination with aggrastat (a glycoprotein [GP] IIb/IIIa inhibitor) at a dose of 10 microg/kg iv. bolus followed by an infusion of 0.15 microg/kg/minute in patients with HIT undergoing percutaneous coronary interventions resulted in elevation of celite activated clotting time (ACT) to 300 seconds followed by a gradual decline and the ACT remained above 200 seconds even after 200 min of drug administration. A bewildering array of newer anticoagulants now exist, such as LMWHs and heparinoids, indirect or direct thrombin inhibitors, oral thrombin inhibitors, such as melagatran (AstraZeneca) and HC-977 (Mitsubishi Pharmaceuticals), Factor IXa inhibitors, indirect or direct Factor Xa inhibitors, Factor VIIa/tissue factor (TF) pathway inhibitor, newer antiplatelet agents, such as GPIIb/IIIa inhibitors, fibrin specific thrombolytic agent, such as tenecteplase and modulation of the endogenous fibrinolytic activity by thrombin activatable fibrinolytic inhibitor (TAFI), Factor XIIIa inhibitors and PAI-1 inhibitors. The quest for newer anticoagulant, antiplatelet and fibrinolytic agents will continue until ideal agents are found.

Journal Article↗
PubMed2000

Abciximab treatment in vitro after aspirin treatment in vivo has additive effects on platelet aggregation, ATP release, and P-selectin expression.

To prevent arterial thrombosis, abciximab is administered together with aspirin. However, whether or not there are benefits to combine abciximab with aspirin is not yet well defined. Healthy volunteers were studied for the effect of aspirin + abciximab using sodium arachidonate and adenosine diphosphate (ADP) alone or in combination to induce platelet activation/aggregation. Abciximab produced complete inhibition of platelet aggregation induced with ADP but only 40% inhibition of aggregation induced by 0.75-mmol/l sodium arachidonate. Abciximab added in vitro to platelet-rich plasma (PRP) from platelets from aspirin-treated donors produced an almost complete inhibition of platelet aggregation. Aspirin, and abciximab alone, did not inhibit adenosine triphosphate (ATP) release as thoroughly as aspirin + abciximab did. Abciximab (3-5 microg/ml) produced inhibition of P-selectin expression induced with 5 (from 46.2 +/- 6.0% to 27.4 +/- 7.0%, P=0.002) and 20-micromol/l ADP (from 53.1 +/- 8.1% to 35.1 +/- 11.0%, P=0.019), but no effect was observed when 0.75-mmol/l sodium arachidonate was used (P=0.721). Aspirin diminished P-selectin expression in sodium arachidonate-stimulated platelets (from 77.7 +/- 11.8% to 40.2 +/- 3.6%, P<0.0001) in non-aspirinated and platelets from aspirin-treated donors, respectively. Abciximab (3, 4, and 5 microg/ml) added to platelets from aspirin-treated donors decreased P-selectin expression in platelets stimulated with sodium arachidonate from 40.2 +/- 8.6% to 25.6 +/- 11.5% (P=0.027), to 20.5 +/- 3.5% (P<0.0001), and to 22.5 +/- 1.8% (P<0.0001). We concluded that the antiplatelet effect of abciximab is greatly increased by aspirin.

Abciximab↗
PubMed2000

Heparinase I acts on a synthetic heparin pentasaccharide corresponding to the antithrombin III binding site.

A synthetic pentasaccharide, containing an intact antithrombin III (ATIII) binding site that is in clinical studies a specific antifactor Xa agent, serves as a substrate for a heparin lyase (heparinase I, EC 4.2.2.7) from Flavobacterium heparinum. Heparinase I, currently being assessed as a heparin reversal agent, also reverses the antifactor Xa activity of this synthetic pentasaccharide by breaking it down to inactive disaccharide and trisaccharide products.

Antithrombin III↗
PubMed2000

Human anti-heparin-platelet factor 4 antibodies are capable of activating primate platelets: towards the development of a HIT model in primates.

In the first step to establish an animal model of heparin-induced thrombocytopenia (HIT) that is physiologically relevant to humans, studies were undertaken to determine the similarities or differences between human and non-human primate (Macaca mulatta) platelets in HIT assay systems. The collagen-, ADP-, and TRAP-induced platelet aggregation, and flow cytometric analysis of P-selectin expression and microparticle formation were similar for both species platelets (p>0.1, n=18 each). The classical HIT assays using platelet-rich plasma (PRP) as well as a flow cytometric assay revealed the activation/aggregation and serotonin release assay (SRA) profiles for both primate and human platelets were similar in response to human HIT positive sera. All assays were heparin concentration-dependent; heparin, at 0.1 U/mL, produced maximum and similar platelet activation/aggregation and SRA responses with both primate (76+/-7%, n=18) and human (68+/-11%, n=20; p>0.1) platelets. At concentrations > or =10 U/mL, heparin suppressed the platelet aggregation and SRA responses in both systems. Primate and human platelets displayed similar behavior to low molecular weight heparin and pentasaccahride in HIT assay systems. Immunoglobulins isolated from serum of patients with HIT caused activation/aggregation of human (65+/-18%, n=10 donors) and primate (79+/-12%, n=6 monkeys, p>0.08) platelets. Unlike human platelets, the primate platelets exhibited a more consistent aggregation/release response (15 out of 18 primate platelets reactive). In contrast, human donors showed wide variations in the activation/release response (4 out of 10 reactive). These observations suggest that primate platelets are activatable by anti-H-PF4 antibodies, and support the hypothesis that primates can be used to develop an animal model to study the pathogenesis of HIT.

Animals↗
PubMed2000

Antithrombin III concentrate in the acute phase of thermal injury.

BACKGROUND: Thermal injury disrupts homeostasis by inducing subclinical disseminated intravascular coagulation, fibrinolysis. and an acquired deficiency of Antithrombin III (ATIII), a natural anticoagulant. As a result, thermally injured patients have a high incidence of hypercoagulability and thrombosis. OBJECTIVE: ATIII (Human) concentrate was given to a thermally injured patient to evaluate safety, and dosage requirements in this setting. DESIGN: The patient was a 40 yr old male with a 68% total burn surface area, right femoral comminuted fracture, and C5-C6 subluxation sustained in a vehicular crash. He received nine infusions of AT III (H) concentrate (100-50 u/kg) within the first four days of injury. RESULT: The ATIII plasma level increased from 45% on admission (normal = 100+/-20%) to 120+/-25% in the next four days. During the 64 day hospitalization, there were 11 grafting procedures with an estimated blood loss (EBL)/procedure: 1140 cc; and EBL/grafted surface area ratio: 0.6 cc cm2. The average time to healing of the meshed autograft was 6.4 days. CONCLUSION: ATIII (H) concentrate can be safely utilized in the acute phase of thermal injury: no excessive bleeding or prolongation of wound healing was documented.

Acute Disease↗
PubMed2000

Statin drugs and dietary isoprenoids as antithrombotic agents.

Statin drugs and various isoprenoids from plant origins inhibit mevalonic acids, cholesterol, and other isoprenoid products. Among these, reduction of farnesyl and geranylgeranyl prenylated proteins impedes signal transduction at the cellular level. The authors envision that limiting such prenylated proteins downregulates thrombin-stimulated events, including decreasing the expression and availability of protease-activated receptor-1 mitigating thrombin stimulation of cells, tissue factor preventing additional thrombin generation, and plasminogen activator inhibitor-1 allowing thrombosis. Additional processes may enhance nitric oxide production and induce other processes. Downregulation of thrombin-stimulated events should promote hypothrombotic or quiescent conditions that reduce cardiovascular disease, thus contributing to longevity.

Animals↗
PubMed2000

Mechanisms of venous and arterial thrombosis in heparin-induced thrombocytopenia.

Since the reports by Weismann and Tobin in 1958 and Roberts et al. in 1964 called attention to paradoxical thrombosis in patients treated with heparin, the thrombotic aspect of the heparin-induced thrombocytopenia syndrome (HIT) has been emphasized. Yet to this day, the mechanism of thrombosis associated with HIT (HITT) is unclear. It is important to understand the etiology of HITT because of its devastating clinical consequences. We believe one rational approach to understand the mechanism underlying HITTS is to invoke Virchow's triad: stasis, vascular injury and a hypercoagulable state. A hypercoagulable state exists in all HIT patients due to platelet activation by heparin antibody binding. Thrombin generation from platelet microparticles and exposed platelet phospholipid, coupled with stasis (elderly bedridden or otherwise sedentary ill patients who comprise the majority of the HIT population), provide two risk factors that can lead to venous thrombosis. A hypercoagulable state coupled with endothelial cell dysfunction due to injury from heparin antibody, activated platelets, leukocytes, platelet microparticles, complement, atherosclerosis or medical intervention can lead to arterial thrombosis. Of patients with HIT, HITT occurs in about 25%, suggesting that a second set of patient specific risk factors, in addition to the generation of pathological heparin antibodies, determine whether HITT will develop. Interaction between activated platelets and other platelets, and with endothelial cells, leukocytes, neutrophils, monocytes and cytokines are areas of research that may provide more specific characterization of the hypercoagulable state and vascular damage. Nuances involving genetic variation in platelets, endothelial cells and immune function are also likely to be a major component of the observed variability of this disease spectrum. Virchow's triad may explain the different manifestations of HITTS.

Anticoagulants↗
PubMed2000

Affinity purification of heparin-dependent antibodies to platelet factor 4 developed in heparin-induced thrombocytopenia: biological characteristics and effects on platelet activation.

Antibodies to heparin platelet factor 4 (H-PF4) complexes were purified from the plasma of three patients with heparin-induced thrombocytopenia (HIT) using affinity chromatography. From each plasma, the largest amount of antibodies was eluted with 2 M NaCl at pH 7.5 (peak 1) and the remainder was obtained using 0.1 M glycine/0. 5 M NaCl at pH 2.5 (peak 2). In an enzyme-linked immunosorbent assay (ELISA), we then showed that each patient had developed antibodies to PF4 displaying different characteristics. In patient 1, peak 1 IgG reacted almost exclusively with H-PF4 complexes, whereas peak 2 IgG had similar reactivity with PF4 whether or not heparin was present. Patient 2 expressed a mixture of IgA, IgM and IgG and both fractions bound to PF4 alone or to H-PF4 complexes. Finally, IgG in patient 3 only bound to H-PF4 and was unreactive with PF4 alone. Using [14C]-serotonin release assays, the antibodies developed in the three patients and exhibiting the strongest ability to activate platelets with heparin were those having the highest affinity to H-PF4. These results strongly support the hypothesis that HIT antibodies to PF4 are heterogeneous regarding their affinity and specificity for target antigens and this may greatly influence their ability to activate platelets and their pathogenicity.

Anticoagulants↗
PubMed2000

Soluble P-selectin in human plasma: effect of anticoagulant matrix and its levels in patients with cardiovascular disorders.

P-Selectin represents a cell surface glycoprotein that is constitutively present in the Weibel-Palade bodies of endothelial cells and in the alpha-granules of platelets. In inflammation and thrombogenic conditions, plasmatic P-selectin levels are markedly elevated, indicating the leakage of this marker from these sites. In this study, a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) utilizing a monoclonal soluble P-selectin (sP-selectin) antibody was employed to assess this marker in blood samples collected in various anticoagulants such as heparin, hirudin, sodium citrate (3.2% and 3.8%), and ethylenediaminetetraacetic acid (EDTA). The soluble P-selectin levels ranged from 26 ng/mL to 44 ng/mL. Sodium citrate (3.8%) was used to collect platelet-poor plasma (PPP) from patients with heparin-induced thrombocytopenia (HIT), coronary angioplasty (CA), or coronary atherectomy (CAT). In comparison with the control group (approximately 30 ng/mL), all of these patient groups showed a marked elevation of sP-selectin levels (HIT = 96 ng/mL [n = 18], CA = 46 ng/mL [n = 6] and CAT = 60 ng/mL [n = 10]). In platelet-rich plasma (PRP) preparations using various anticoagulants, the sP-selectin levels were markedly higher, ranging from 87 ng/ mL to 117 ng/mL (n = 10). In patients recruited into a clinical trial (the argatroban [ARG] 911 Study), in which argatroban was used as an alternate anticoagulant in patients with HIT, a 25% to 35% decrease in sP-selectin levels was observed after 72 hours of argatroban treatment. In addition, the relative ratio between levels in PRP and PPP in these patients differed, suggesting that the anticoagulant matrix influences the sP-selectin levels. These data clearly suggest that the anticoagulant matrix and blood collection procedures may significantly influence the plasmatic P-selectin levels. Furthermore, in different clinical conditions, elevation of this marker may reflect endogenous platelet activation; however, optimal anticoagulant for blood collection is important for proper diagnostic validation.

Adult↗