Roger C. Carroll

Early evaluation of acute traumatic coagulopathy by thrombelastography

Posttraumatic coagulopathy is a major cause of morbidity. This prospective study evaluated the thrombelastography (TEG) system and PlateletMapping (Haemoscope Corporation, Niles, Ill) values posttrauma, and it correlated those values with transfusions and fatalities. After institutional review board approval, assays were performed on 161 trauma patients. One citrated blood sample was collected onsite (OS), and 1 citrate and 1 heparinized sample were collected within 1 h of arrival to the emergency department (ED). Paired and unpaired t-testing was performed for nominal data with chi square testing for categorical values. Except for a slight increase in clot strength (maximal amplitude (MA)), there were no significant changes from OS to the ED. None of the TEG parameters were significantly different for the 22 patients who required transfusion. PlateletMapping showed lower platelet adenosine diphosphate (ADP) responsiveness in patients who needed transfusions (MA = 22.7 +/- 17.1 vs MA = 35.7 +/- 19.3, P = 0.004) and a correlation of fibrinogen <100 mg/dL with fatalities (P = 0.013). For the 14 fatalities, TEG reaction (R) time was 3703 +/- 11,618 versus 270 +/- 393 s (P = < 0.001), and MA was 46.4 +/- 22.4 versus 64.7 +/- 9.8 mm (P < 0.001). Hyperfibrinolysis (percent fibrinolysis after 60 min (LY60) >15%) was observed in 3 patients in the ED with a 67% fatality rate (P = < 0.001 by chi-square testing). PlateletMapping assays correlated with the need for blood transfusion. The abnormal TEG System parameters correlated with fatality. These coagulopathies were already evident OS. The TEG assays can assess coagulopathy, platelet dysfunction, and hyperfibrinolysis at an early stage posttrauma and suggest more effective interventions.

Measurement of functional fibrinogen levels using the Thrombelastograph

STUDY OBJECTIVEnTo validate a Thromboelastograph (Haemoscope Corporation, Niles, IL) assay for functional fibrinogen.nnnDESIGNnCorrelation study of the Thromboelastograph assay with two conventional fibrinogen assays by the standard Clauss method.nnnSETTINGnResearch laboratory of a university medical center.nnnPARTICIPANTS AND INTERVENTIONSnBlood samples were obtained from 19 healthy volunteers.nnnMEASUREMENT AND MAIN RESULTSnThromboelastograph assays, using heparinized whole blood from 19 healthy donors, indicated that reptilase-XIIIa mixture (Activatorf)-generated clot shear elasticity in dynes per square centimeter (Gf) correlated with fibrinogen (mg/dL). Blood from four donors was used to define the contribution of hematocrit (Hct) to Gf by titration with platelet-rich plasma. The Gf versus Hct gave linear correlations (r2 = 0.746) with Gf = 1258 - 17.8 x % Hct. A commercial collection of 19 normal, 10 borderline, and one deficient for functional fibrinogen-citrated plasmas was assayed for Gf after recalcification using Activatorf. Of the 30 plasma samples, four were from factor X- or factor VII-deficient donors and one was from a coumadin-treated donor. There was a linear correlation of Activatorf Gf with functional fibrinogen (r2 = 0.940) with Gf = -730 + 9.21 x fibrinogen (mg/dL).nnnCONCLUSIONnThrombelastography with Activatorf may be used to determine fibrinogen levels in whole blood.

A novel thrombelastograph® tissue factor/kaolin assay of activated clotting times for monitoring heparin anticoagulation during cardiopulmonary bypass

We used a thrombelastograph (TEG®) assay with tissue factor and kaolin (TEG® TF/K) to measure activated clotting time (ACT) in 31 patients during cardiopulmonary bypass. For comparison, ACTs were also determined by a Hemochron Jr. Signature® and a Hepcon® HMS. The TEG® TF/K correlated with both the Hepcon (r2 = 0.789) and Hemochron (r2 = 0.743) ACTs. The average ACT after heparin was 319 ± 119 s (mean ± SD) for the TEG® TF/K compared with 624 ± 118 s for the Hepcon instrument. To evaluate the effects of hemodilution on TEG® TF/K and Hemochron assays, ACT assays were performed on blood diluted to 50% and titrated with heparin from 0 to 6 U/mL. Both instruments showed significant (P < 0.01) changes in the ACT-versus-heparin slope, but the 0 heparin intercept for the TEG® TF/K ACTs was not significantly changed (P = 0.292), in contrast to that for the Hemochron device (P = 0.041). Both instruments also indicated the same 1.3:1 ratio of protamine to heparin for optimum heparin neutralization, with increasing ACTs at ratios >2.6:1. The TEG® TF/K ACT assay rapidly monitors heparin anticoagulation, in addition to the capabilities of this instrument to monitor platelet function, clotting factors, and fibrinolysis.

Blocking platelet aggregation inhibits thromboxane A2 formation by low dose agonists but does not inhibit phosphorylation and activation of cytosolic phospholipase A2.

Inhibition of aggregation by Ro 44-9883, a potent and selective non-peptide GPIIb/IIIa antagonist, resulted in inhibition of serotonin secretion induced by weak agonists such as ADP or low doses of either thrombin receptor agonist peptide (TRAP) or collagen. In contrast, alpha granule secretion was inhibited to different extents dependent on donor, averaging 60% inhibition. Inhibition of serotonin secretion correlated with an inhibition of thromboxane A2 (TxA2) formation, both of which were overcome by higher doses of TRAP or collagen. Ro 44-9883 had no effect on the already reduced serotonin secretion and TxA2 formation in Glanzmanns thrombasthenic platelets. Restoration of serotonin secretion in the absence of aggregation requires both TxA2 and lysophosphatidic acid. In addition, Ro 44-9883 inhibition of TxA2 formation was not due to a lack of phospholipase A2 (PLA2) phosphorylation and activation as assayed in vitro. These results suggest that aggregation is required for weak or low dose agonist induced in vivo activity of PLA2, possibly by either regulating phospholipid substrate availability or interaction of PLA2 with platelet membranes.

Lotrafiban: An oral platelet glycoprotein IIb/IIIa blocker

Platelets play a major role in thrombus formation, as well as in the pathogenesis of atherothrombosis. Inhibition of platelet function is now emphasised more than ever for prevention and treatment of almost all vascular diseases, since thrombosis is established as the key pathogenic event causing acute ischaemic coronary and cerebrovascular syndromes. Although acetylsalicylic acid (aspirin) has been shown to reduce the incidence of myocardial infarction and stroke, its effect is weak and more effective antithrombotic agents are required to manage patients at high-risk for recurrent vascular events. Platelet glycoprotein IIb/IIIa receptor (GPIIb/IIIa) blockade represents a significant advance in interventional cardiology and treatment of acute ischaemic syndromes. The past several years have seen the introduction of many platelet GPIIb/IIIa blockers into the clinical arena targeting the unique platelet GPIIb/IIIa receptor for the adhesive proteins, fibrinogen and von Willebrand Factor. Platelet GPIIb/IIIa blockers administered intravenously have proven efficacious in mitigating arterial thrombosis in acute coronary syndromes (unstable angina and non-ST-elevation myocardial infarction) and percutaneous coronary interventions (PCI) such as balloon dilatation and stent implantation. Currently, orally-active platelet GPIIb/IIIa blockers are being developed to provide additional benefits for primary and secondary prevention of thrombosis as chronic treatment, especially in high-risk patients. Lotrafiban (SmithKline Beecham) is a member of the latest generation of orally-active platelet GPIIb/IIIa blockers undergoing Phase III clinical trials to test the relative effectiveness versus other oral platelet inhibitors for ischaemic conditions including unstable angina, restenosis after PCI and stroke. Lotrafiban is converted from an esterified prodrug by plasma and liver esterases to a peptidomimetic of the arginine-glycine-aspartic acid amino acid sequence. This sequence itself mimics the binding site of fibrinogen and von Willebrand Factor to the platelet GPIIb/IIIa receptor. Preliminary results of the clinical trial APLAUD (antiplatelet useful dose) show that lotrafiban is clinically safe and well-tolerated in patients with recent myocardial infarction, unstable angina, transient ischaemic attack (TIA), or stroke when added to aspirin therapy. With lotrafiban, a worldwide large-scale Phase III clinical trial BRAVO (blockage of the GPIIb/IIIa receptor to avoid vascular occlusion) is currently underway. In general, GPIIb/IIIa blockade seems clinically very promising. A number of unresolved issues, however, remain to be elucidated.

Post interventional cardiology urinary thromboxane correlates with PlateletMapping® detected aspirin resistance

INTRODUCTIONnWe have previously defined aspirin resistance detected by TEG PlateletMapping using arachidonic acid (AA). This aspirin resistance is observed as platelet activation (>20%) by AA in whole blood, even though the isolated platelets are inhibited by aspirin. This platelet activation in whole blood is due to a transcellular pathway mediated by platelets and leukocytes.nnnMETHODSnTo determine if this PlateletMapping assay of aspirin resistance on pre-procedure blood samples correlated with an in vivo response we assayed the first voided urine samples collected 2-8 hours post interventional cardiology procedures for 11-dehydro thromboxane B2.nnnRESULTS AND CONCLUSIONSnWe detected 27 aspirin resistant patients out of a total of 81 (33%), in agreement with our previous study. All of these patients were on aspirin therapy, confirmed by a <20% aggregation response to AA by light transmission platelet aggregometry using isolated platelet rich plasma. Aspirin resistant patients urine samples (14 out of a total of 60 patients analyzed) contained significantly (P=0.008) higher 11-dehydro thromboxane B2 levels than the other 46 aspirin sensitive patients urine samples. Since our previous study implicated 12- and 15-lipoxygenases in this pathway, we also assayed for polymorphisms to determine any correlation with aspirin resistance. A correlation was found in a polymorphism affecting the lipoxygenase domain of platelet 12-lipoxygenase. This result indicates that aspirin resistance detected in whole blood by the TEG PlateletMapping assay correlates with a physiological consequence in terms of thromboxane formation. This is the first report of such a correlation.

The effect of tetradecanoylphorbol acetate on calcium-ion mobilization, protein phosphorylation and cytoskeletal assembly induced by thrombin or arachidonate

Tetradecanoylphorbol acetate (TPA) activates primarily only the protein kinase C pathway not the calcium ion-dependent pathway in platelets. The net effect of this split activation is that only the pseudopodal cytoskeleton assembles, not the contractile cytoskeleton needed for rapid secretion. In this study, platelets were first activated with TPA, then activated secondarily with either thrombin or arachidonate and the subsequent dense body secretion, calcium-ion mobilization, protein phosphorylation and cytoskeletal assembly compared to these same processes in control platelets activated solely with either thrombin or arachidonate. Secretion was reduced as the length of time between the primary and secondary activation was increased; but at a 2-3 min interval, where the activation by TPA was essentially complete, the reduction in the total radiolabeled serotonin secreted was small. Furthermore, nearly normal cytosolic calcium-ion increases, phosphorylation of myosin light chain and contractile cytoskeletal development were induced by thrombin or arachidonate after this interval. Prior treatment of the platelets with 100 microM acetylsalicylate to block the cyclooxygenase-dependent pathway caused minor reduction in dense-body secretion induced by TPA or thrombin or the combination of both, but otherwise the relative results were comparable to the untreated platelets. Therefore, short-term prior activation of gel-filtered platelets with TPA, even at concentrations in excess of 100-times that required to saturate protein kinase C, does not prevent normal activation of the calcium ion dependent processes through either the cyclooxygenase-dependent or -independent pathway. Longer-term preincubations with TPA differentially inhibit the secretion response induced by thrombin and arachidonate.

Thromboelastographic changes in patients experiencing an acute ischemic stroke and receiving alteplase.

BACKGROUNDnThromboelastography is a method of measuring whole-blood coagulation changes and has been used to guide therapy and monitor changes in a variety of disease states. However, few studies have investigated the thromboelastographic changes experienced in a patient who has received alteplase for an acute ischemic stroke. This pilot study sought to describe the effect of alteplase on the thromboelastogram tracings of patients experiencing an acute ischemic stroke.nnnMETHODSnThis was an institutional review board-approved prospective cohort study. Patients who presented to the emergency department with symptoms of acute ischemic stroke and received intravenous alteplase were evaluated for inclusion. Blood samples were obtained before alteplase administration and at 30, 60, 90, 120, and 150xa0minutes after alteplase administration. In addition, baseline variables collected included patient age, sex, prothrombin time, partial thromboplastin time, and the use of pretreatment anticoagulants or antiplatelet agents. Patients were also followed throughout their hospital stay for development of intracranial hemorrhage.nnnRESULTSnA total of 7 patients were included in the analysis. At baseline, thromboelastogram parameters of all patients were within the normal range. The maximum inhibition of fibrin buildup was seen at 30xa0minutes after the start of alteplase infusion, and the lowest clot strength was observed at 60xa0minutes after initiation of alteplase. Most patients return to near baseline parameters within 150xa0minutes of alteplase initiation; however, 2 patients did not return to their baseline values within the 150-minute time frame.nnnCONCLUSIONSnOur study suggests that thromboelastogram (TEG) is a useful tool for determining changes in the coagulation system of patients whom have received recombinant tissue plasminogen activator (rt-PA). Further study is needed to determine if TEG can be used to predict those patients who may be at higher risk of adverse events because of rt-PA.

A Comparison of VerifyNow® with PlateletMapping® -Detected Aspirin Resistance and Correlation with Urinary Thromboxane

BACKGROUND:Aspirin-resistant platelet activation in whole blood is attributable to a transcellular pathway not detected by isolated platelet aggregometry. Aspirin resistance as defined by urinary thromboxane levels is associated with increased risk for myocardial infarction or cardiac death. Whole blood point-of-care assays may also detect aspirin resistance. METHODS:We compared PlateletMapping® with VerifyNow® for detecting aspirin resistance in 200 patients undergoing invasive cardiac procedures. This included 10 patients not receiving aspirin therapy for comparison. The assay results were correlated with urinary 11-dehydro-thromboxane B2 collected 2 to 8 hours after the procedure. RESULTS:PlateletMapping detected aspirin resistance in 32% of patients. VerifyNow detected aspirin resistance in 6% of patients. A patient’s compliance with aspirin therapy was confirmed by a <20% aggregation response to arachidonic acid by light transmission aggregometry. Aspirin-resistant patients as determined by PlateletMapping had significantly (P < 0.001) higher urinary 11-dehydro-thromboxane B2 levels than aspirin-sensitive patients but significantly (P = 0.001) lower levels than patients not receiving aspirin therapy. There was no significant difference in urinary 11-dehydro-thromboxane B2 for aspirin-resistant compared with aspirin-sensitive patients as determined by VerifyNow, but the confidence intervals were wide. There was no significant correlation of resistance as defined by PlateletMapping with aspirin dose. However, there was significant increased aspirin sensitivity with clopidogrel (0.0006) or statin (0.004) cotherapies. There also was a significant correlation of smoking with aspirin resistance. CONCLUSIONS:These results indicate that PlateletMapping could be a useful point-of-care assay to identify aspirin-resistant patients for better perioperative risk stratification and management.

Synergistic Outside-In Regulation of Platelet Activation by GPIIb/IIIa Ligand-Induced Conformation and Oligomerization

Full platelet activation with serotonin secretion and thromboxane A(2) (TxA(2)) formation induced by a low dose of thrombin receptor agonist peptide (TRAP) or high dose ADP requires platelet aggregation. This requirement can be replaced by pretreatment of platelets with a combination of reagents including: GPIIb/IIIa inhibitors yielding ligand-induced binding sites (LIBS), either arginine-glycine-aspartate-serine (RGDS) peptide or Ro 43-5054, cytochalasin to disrupt actin filaments and crosslinking by a GPIIb/IIIa mAb (pl-62). Crosslinking is required since Fab fragments of pl-62 do not support activation. Engagement of the Fc receptor by the mAb Fc domain is not required for pl-62 augmentation, since it is not blocked by the anti-Fc receptor mAb, IV-3. Another GPIIb/IIIa inhibitor, Ro 44-9883, not yielding LIBS epitopes, serves as a negative control and shows a requirement for LIBS in addition to crosslinking. Focal adhesion kinase tyrosine phosphorylation induced by TRAP is blocked by these GPIIb/IIIa antagonists, but restored by pl-62 crosslinking independent of LIBS induction. Tyrosine phosphorylation of a peptide comigrating with p38 MAP kinase is also inhibited by these antagonists and restored by pl-62 crosslinking. However, p38 MAP kinase activation by low dose TRAP is not affected by these aggregation inhibitors. Tyrosine phosphorylation of a 34-kDa phosphoprotein in the absence of aggregation or TxA(2) formation was uniquely augmented by Ro 43-5054 but not Ro 44-9883 under the above activation conditions.