New interest in fibrinogen as an ischemic risk factor.

Platelet-fibrin clot formation is a key process in acute arterial thrombosis. The relationship between thrombin-induced platelet-fibrin clot strength (P-FCS) and fibrinogen levels in patients with cardiovascular disease (CVD) and COVID-19 has not been studied. In thhe current study, the contribution of fibrinogen to P-FCS has been explored in healthy subjects (n=157), patients hospitalized with COVID-19 (n=116), and patients with CVD (n=93) using thrombelastography (TEG 6s) with citrate cartridge. We found that thrombin-induced P-FCS, fibrin clot strength (F-CS) and fibrinogen levels (FLEV) were higher among patients with CVD and COVID-19 compared to HS (p<0,05 for all) and highest among patients with COVID-19. P-FCS, an established risk factor for post-PCI ischemic event occurrences, was associated with both F-CS and FLEV (R2=0.67, p<0.001 for both comparisons. These data indicate that fibrinogen levels strongly influence the viscoelastic strength of the platelet-fibrin clot, fibrinogen may be an important driving factor for arterial thrombosis in the presence of potent platelet inhibition and may be as equally important a risk factor as high platelet reactivity. Since P-FCS is significantly associated with fibrinogen levels, the role of fibrinogen as a risk factor for arterial ischemic event occurrences should be further studied to improve antithrombotic therapy personalization.

Early assessment of the pharmacokinetic and pharmacodynamic effects following acetylsalicylic acid loading: toward a definition for acute therapeutic response.

Despite decades of investigations, the optimal assessment of the "therapeutic response" to early after loading dose of acetylsalicylic acid (ASA) remains unclear. Limited information is available on the relation between pharmacodynamic (PD) and pharmacokinetic (PK) measurements assessed immediately after ASA administration. Serial PD and PK analyses were performed immediately after a single 162 or 650 mg dose of chewed and swallowed ASA in ten healthy adults. ASA response was defined as > 95% inhibition of serum thromboxane (Tx)B2, < 550 aspirin reaction units (ARU) by VerifyNow Aspirin (VN) test, and ≤ 20% arachidonic acid (AA)-induced platelet aggregation (PA). Correlation analyses between PK and PD measurements and receiver operating characteristic (ROC) curve analyses were performed. ASA response measured by VN test and AA-induced PA was achieved within 30 min of ASA administration. A correlation was observed between ARU and AA-induced maximum PA (r = 0.69, p < 0.001), serum TxB2 (r = 0.74 and p < 0.001), and serum TxB2 inhibition (r = 0.79, p < 0.001). In ROC curve analyses, ≤ 558 ARU and ≤ 7% AA-induced PA were associated with > 95% inhibition of TxB2. 686 ng/ml plasma ASA cut-off point was associated with > 95% inhibition of serum TxB2, ≤ 7% 1 mM AA-induced PA, and ≤ 585 ARU. A modest ~ 50% inhibition of TxB2 inhibition was associated with marked inhibition of 1 mM AA-induced platelet aggregation by LTA. Our analyses demonstrated important relationships between pharmacodynamic, and pharmacokinetic parameters measured immediately following oral ASA and cutoff values for ARU and AA-induced PA that is associated with > 95% inhibition of serum TxB2.

Development of a routine bedside CYP2C19 genotype assessment program for antiplatelet therapy guidance in a community hospital catheterization laboratory.

Genotype based personalized antiplatelet therapy in the setting of percutaneous coronary intervention (PCI) has been studied in clinical trials. Despite the demonstrated risk associated with CYP2C19 loss-of-function (LoF) carriage in clopidogrel-treated PCI patients, real-world implementation of genotyping for PCI has been low. The goal of the current study was to provide CYP2C19 genotype information to the interventionalist prior to the completion of the catheterization to facilitate immediate personalized antiplatelet therapy. Routine personalization of P2Y12 inhibitor therapy for PCI in a community hospital cardiac catheterization laboratory by POC genotyping with the SpartanRx system was first offered in February 2017. A best practice advisory (BPA) based on the Clinical Pharmacogenetics Implementation Consortium Guideline for CYP2C19 genotype and clopidogrel therapy was placed in the electronic health record prescription medication ordering system. By December 2019, 1,052 patients had CYP2C19 genotype testing, 429 patients underwent PCI with genotype guided antiplatelet therapy, and 250 patients underwent PCI without genotype testing and received antiplatelet therapy at the discretion of the treating physician. BPA compliance was 93. 87% of LoF allele carriers were prescribed ticagrelor or prasugrel whereas 96% of non-LoF allele carriers were prescribed clopidogrel. The genotyping results were available within 1 h and made immediately available for decision making by the interventional cardiologist. POC CYP2C19 genotyping is feasible in a community hospital catheterization laboratory and is associated with high rate of best practice compliance.Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT03040622.

Utility of VerifyNow to assess the immediate pharmacodynamic response of chewed and swallowed aspirin: comparison with aggregometry.

VerifyNow (VN) test is a less laborious method to assess pharmacodynamics (PD) compared to light transmittance aggregometry (LTA). VN assay has not been used to study the immediate PD effects of acetylsalicylic acid (ASA). Ten healthy volunteers were randomly assigned to a single 162 or 650 mg dose of chewed and swallowed ASA. Pharmacodynamic and pharmacokinetic measurements were performed at baseline and serially up to 60 min after ASA administration. Onset by VN was 20 ± 7 min with 162 mg and 13 ± 7 min with 650 mg ASA (p = .07). Onset by 1 mM AA-induced PA was 13 ± 12 min with 162 mg and 7 ± 3 min with 650 mg ASA (p=NS). VN correlated with AA-induced PA (r = 0.80, p < .001) and serum TxB2 levels (r = 0.76, p < .001). 95% inhibition of serum TxB2 was achieved at 38 ± 22 min and 22 ± 8 min with the 162 and 650 mg ASA, respectively (p = .08). The onset and extent of the antiplatelet effect of 650 mg ASA is numerically faster and greater than the 162 mg dose. VN identifies the onset, extent, and dose response to ASA therapy. The ease of using VN should facilitate multicenter PD investigations of ASA.

Aspirin (acetylsalicylic acid) is an important drug widely used to prevent adverse ischemic events in patients with cardiovascular disease. Platelet aggregation and thromboxane B₂ levels in blood samples by complex laboratory methods are used to assess platelet response to aspirin. VerifyNow assay is a simple laboratory test that has not been used to assess the immediate effect of aspirin. In this study, conducted in 10 healthy volunteers, we compared the immediate platelet response to aspirin by serially assessing platelet aggregation by aggregometry and VerifyNow assay, and thromboxane B₂ levels. We also measured plasma levels of acetylsalicylic acid and salicylic acid. Our study demonstrated that the VerifyNow Aspirin test identifies the onset, extent, and dose-response to aspirin therapy. The ease of using the VerifyNow test should facilitate multicenter pharmacodynamic investigations of aspirin.

Prognostic impact of hypercoagulability and impaired fibrinolysis in acute myocardial infarction.

AIMS: Atherothrombotic events are influenced by systemic hypercoagulability and fibrinolytic activity. The present study evaluated thrombogenicity indices and their prognostic implications according to disease acuity. METHODS AND RESULTS: From the consecutive patients undergoing percutaneous coronary intervention (PCI), those with thrombogenicity indices (n = 2705) were grouped according to disease acuity [acute myocardial infarction (AMI) vs. non-AMI]. Thrombogenicity indices were measured by thromboelastography (TEG). Blood samples for TEG were obtained immediately after insertion of the PCI sheath, and TEG tracing was performed within 4 h post-sampling. Major adverse cardiovascular events (MACE, a composite of cardiovascular death, non-fatal myocardial infarction, and non-fatal stroke) were evaluated for up to 4 years. Compared with non-AMI patients, AMI patients had higher platelet-fibrin clot strength [maximal amplitude (MA): 66.5 ± 7.8 vs. 65.3 ± 7.2 mm, P < 0.001] and lower fibrinolytic activity [clot lysis at 30 min (LY30): 0.9 ± 1.8% vs. 1.1 ± 1.9%, P < 0.001]. Index AMI presentation was associated with MA [per one-mm increase: odds ratio (OR): 1.024; 95% confidence interval (CI): 1.013-1.036; P < 0.001] and LY30 (per one% increase: OR: 0.934; 95% CI: 0.893-0.978; P = 0.004). The presence of high platelet-fibrin clot strength (MA ≥68 mm) and low fibrinolytic activity (LY30 < 0.2%) was synergistically associated with MACE occurrence. In the multivariable analysis, the combined phenotype of 'MA ≥ 68 mm' and 'LY30 < 0.2%' was a major predictor of post-PCI MACE in the AMI group [adjusted hazard ratio (HR): 1.744; 95% CI: 1.135-2.679; P = 0.011], but not in the non-AMI group (adjusted HR: 1.031; 95% CI: 0.499-2.129; P = 0.935). CONCLUSION: AMI occurrence is significantly associated with hypercoagulability and impaired fibrinolysis. Their combined phenotype increases the risk of post-PCI atherothrombotic event only in AMI patients. These observations may support individualized therapy that targets thrombogenicity for better outcomes in patients with AMI. CLINICAL TRIAL REGISTRATION: Gyeongsang National University Hospital (G-NUH) Registry, NCT04650529.

Deficiency of MMP1a (Matrix Metalloprotease 1a) Collagenase Suppresses Development of Atherosclerosis in Mice: Translational Implications for Human Coronary Artery Disease.

[Figure: see text].

Metformin use in patients hospitalized with COVID-19: lower inflammation, oxidative stress, and thrombotic risk markers and better clinical outcomes.

Diabetes mellitus (DM) is associated with a greater risk of COVID-19 and an increased mortality when the disease is contracted. Metformin use in patients with DM is associated with less COVID-19-related mortality, but the underlying mechanism behind this association remains unclear. Our aim was to explore the effects of metformin on markers of inflammation, oxidative stress, and hypercoagulability, and on clinical outcomes. Patients with DM on metformin (n = 34) and metformin naïve (n = 41), and patients without DM (n = 73) were enrolled within 48 h of hospital admission for COVID-19. Patients on metformin compared to naïve patients had a lower white blood cell count (p = 0.02), d-dimer (p = 0.04), urinary 11-dehydro thromboxane B2 (p = 0.01) and urinary liver-type fatty acid binding protein (p = 0.03) levels and had lower sequential organ failure assessment score (p = 0.002), and intubation rate (p = 0.03), fewer hospitalized days (p = 0.13), lower in-hospital mortality (p = 0.12) and lower mortality plus nonfatal thrombotic event occurrences (p = 0.10). Patients on metformin had similar clinical outcomes compared to patients without DM. In a multiple regression analysis, metformin use was associated with less days in hospital and lower intubation rate. In conclusion, metformin treatment in COVID-19 patients with DM was associated with lower markers of inflammation, renal ischemia, and thrombosis, and fewer hospitalized days and intubation requirement. Further focused studies are required to support these findings.

Serial assessment of thrombogenicity and hemodynamics in patients with type II diabetes in a clinical research unit: Evidence for circadian variations in clot formation.

BACKGROUND: Circadian fluctuations in thrombogenicity and hemostasis play a role in acute cardiovascular thrombotic events occurring in the early morning hours. There is a lack of data assessing thrombogenicity, platelet function, and hemodynamics to investigate diurnal variations in a high cardiovascular risk population. METHODS: This was an exploratory, single-center study conducted in aspirin-treated patients with Type II Diabetes Mellitus (T2DM) (n = 37) with documented vascular disease and/or multiple cardiovascular risk factors. Hemodynamic monitoring and blood sample collection for thromboelastography (TEG) and platelet function testing were done serially at 7-9 AM (morning), 7-9 PM (evening), 11 PM-1 AM (night), and at 5-7 AM (awakening). RESULTS: R-value measured by TEG was shorter during awakening hours than during the night and day hours (p < 0.05). There were no changes in platelet reactivity in response to arachidonic acid, adenosine diphosphate, and collagen between time points. Pulse pressure (PP) was highest during awakening hours (p < 0.05). CONCLUSION: Study findings provide a mechanistic explanation for increased thrombotic events observed in the early waking hours among diabetics with multiple cardiovascular risk factors. The role of chronotherapy in reducing coagulability and PP to improve clinical outcomes should be explored.

The role of viscoelastic testing in assessing peri-interventional platelet function and coagulation.

We carried out a literature search in MEDLINE (PubMed) and EMBASE literature databases to provide a concise review of the role of viscoelastic testing in assessing peri-interventional platelet function and coagulation. The search identified 130 articles that were relevant for the review, covering the basic science of VHA and VHA in clinical settings including cardiac surgery, cardiology, neurology, trauma, non-cardiac surgery, obstetrics, liver disease, and COVID-19. Evidence from these articles is used to describe the important role of VHAs and platelet function testing in various peri-interventional setups. VHAs can help us to comprehensively assess the contribution of platelets and coagulation dynamics to clotting at the site-of-care much faster than standard laboratory measures. In addition to standard coagulation tests, VHAs are beneficial in reducing allogeneic transfusion requirements and bleeding, in predicting ischemic events, and improving outcomes in several peri-interventional care settings. Further focused studies are needed to confirm their utility in the peri-interventional case.

PAR1 (Protease-Activated Receptor 1) Pepducin Therapy Targeting Myocardial Necrosis in Coronary Artery Disease and Acute Coronary Syndrome Patients Undergoing Cardiac Catheterization: A Randomized, Placebo-Controlled, Phase 2 Study.

OBJECTIVE: Arterial thrombosis leading to ischemic injury worsens the prognosis of many patients with cardiovascular disease. PZ-128 is a first-in-class pepducin that reversibly inhibits PAR1 (protease-activated receptor 1) on platelets and other vascular cells by targeting the intracellular surface of the receptor. The TRIP-PCI (Thrombin Receptor Inhibitory Pepducin in Percutaneous Coronary Intervention) trial was conducted to assess the safety and efficacy of PZ-128 in patients undergoing cardiac catheterization with intent to perform percutaneous coronary intervention. Approach and Results: In this randomized, double-blind, placebo-controlled, phase 2 trial, 100 patients were randomly assigned (2:1) to receive PZ-128 (0.3 or 0.5 mg/kg), or placebo in a 2-hour infusion initiated just before the start of cardiac catheterization, on top of standard oral antiplatelet therapy. Rates of the primary end point of bleeding were not different between the combined PZ-128 doses (1.6%, 1/62) and placebo group (0%, 0/35). The secondary end points of major adverse coronary events at 30 and 90 days did not significantly differ but were numerically lower in the PZ-128 groups (0% and 2% in the PZ-128 groups, 6% and 6% with placebo, p=0.13, p=0.29, respectively). In the subgroup of patients with elevated baseline cardiac troponin I, the exploratory end point of 30-day major adverse coronary events + myocardial injury showed 83% events in the placebo group versus 31% events in the combined PZ-128 drug groups, an adjusted relative risk of 0.14 (95% CI, 0.02-0.75); P=0.02. CONCLUSIONS: In this first-in-patient experience, PZ-128 added to standard antiplatelet therapy appeared to be safe, well tolerated, and potentially reduced periprocedural myonecrosis, thus providing the basis for further clinical trials. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02561000.

Defining platelet response to acetylsalicylic acid: the relation between inhibition of serum thromboxane B2 and agonist-induced platelet aggregation.

Arachidonic acid (AA)-induced platelet aggregation (PA) and serum thromboxane B2 (TxB2) inhibition are widely used to indicate cyclooxygenase-1 activity and the antiplatelet effect of acetylsalicylic acid (ASA). Despite decades of investigations, the relation between these measurements remains unclear. We sought to evaluate the relation between AA-PA and serum TxB2 inhibition. We serially measured AA-PA (conventional aggregation), serum TxB2, plasma ASA and salicylic acid (SA) (liquid chromatography-mass spectrometry), and urinary 11-dehydro thromboxane B2 (u11-dh TxB2) (enzyme-linked immunosorbent assay) levels at 10 times over 24 hours in seventeen healthy volunteers receiving a single dose of 162 mg chewed and swallowed ASA (n = 6), 50 mg inhaled ASA (n = 6), or 100 mg inhaled ASA (n = 5) (ClinicalTrials.gov Identifier: NCT04328883, April 1, 2020). Baseline variability was more pronounced with serum TxB2 (31-680 ng/mL) as compared to maximal AA-PA (65-81%) and u11-dh TxB2 (1556-4440 pg/mg creatinine). The relation between serum TxB2 inhibition and AA-PA was stepwise; after 30-40% inhibition of serum TxB2, AA-PA fell to < 5%. By receiver operating characteristic curve analysis using AA-PA < 5% to define aspirin responsiveness, serum TxB2 inhibition > 49% and u11-dh TxB2 < 1520 pg/mg creatinine met the definition. Our study demonstrates a non-linear relation between serum TxB2 inhibition and AA-PA. Aggregation was nil once TxB2 inhibition reached > 49%. Moreover, these results suggest that the definition of > 95% inhibition of serum TxB2 to indicate the level of platelet COX-1 inhibition needed for clinical efficacy may be overestimated and should be re-considered in future translational research investigations that attempt to link the clinical efficacy of ASA with a laboratory measurement cutoff.

Clinical validation of AggreGuide A-100 ADP, a novel assay for assessing the antiplatelet effect of oral P2Y12 antagonists.

In this prospective, 3-arm, repeated-measure multicenter investigation in 280 patients with cardiovascular risk factors, platelet aggregation was measured with the novel AggreGuide A-100 ADP (A-100 ADP) and VerifyNow (VN)-PRU assays at baseline, and after United States Food and Drug Administration approved loading and 7 days maintenance doses of clopidogrel (n = 94), prasugrel (n = 43) or ticagrelor, (n = 143). Based on the predetermined cutoff values of < 4.7 platelet activity index with A-100 ADP assay to indicate antiplatelet response, more than 91% of patients met the criteria following loading and maintenance doses of prasugrel and more than 84% patients met the criteria following loading and maintenance doses of ticagrelor whereas only 32% and 51% of patients met the criteria following loading and maintenance doses of clopidogrel, respectively. The total percent agreement between the A-100 ADP and VN-PRU assays was 89%. The A-100 ADP assay, which includes whole blood in motion, performs comparably to the VN-PRU assay in a study of patients with cardiovascular risk factors treated with P2Y12 inhibitors possessing known differences in antiplatelet potencies. Trial registration ClinicalTrials.gov Identifier: NCT3111420.

Direct oral anticoagulants: a review on the current role and scope of reversal agents.

New guideline recommendations prefer direct oral anticoagulants (DOACs) over warfarin in DOAC-eligible patients with atrial fibrillation and patients with venous thromboembolism. As expected with all antithrombotic agents, there is an associated increased risk of bleeding complications in patients receiving DOACs that can be attributed to the DOAC itself, or other issues such as acute trauma, invasive procedures, or underlying comorbidities. For the majority of severe bleeding events, the widespread approach is to withdraw the DOAC, then provide supportive measures and "watchful waiting" with the expectation that the bleeding event will resolve with time. However, urgent reversal of anticoagulation may be advantageous in patients with serious or life-threatening bleeding or in those requiring urgent surgery or procedures. Until recently, the lack of specific reversal agents, has affected the uptake of these agents in clinical practice despite a safer profile compared to warfarin in clinical trials. In cases of life-threatening or uncontrolled bleeding or when patients require emergency surgery or urgent procedures, idarucizumab has been recently approved for reversal of anticoagulation in dabigatran-treated patients and andexanet alfa for factor Xa inhibitor-treated treated patients. The current review summarizes the current clinical evidence and scope of these agents with the potential impact on DOAC use in clinical practice.

Detailed thrombogenicity phenotyping and 1 year outcomes in patients undergoing WATCHMAN implantation: (TARGET-WATCHMAN) a case-control study.

The relation of device related thrombosis (DRT) and major bleeding after left atrial appendage closure (LAAC) to laboratory thrombosis and hemostasis markers has not been studied. We performed a prospective case control study to identify clinical characteristics and laboratory markers in patients who developed DRT and major bleeding following WATCHMAN LAAC. Thromboelastography, platelet aggregation (PA), urinary 11-dehydrothromboxane B2 (UTX), fibrinogen, D-dimer, thrombin time and von Willebrand factor activity were determined at baseline, immediately following, and at 45 and 180 days post-LAAC (n = 32) and outcomes were followed for 1 year. Baseline characteristics and thrombogenic profiles of patients with and without DRT and/or BARC bleeding were compared. Mean age was 76 ± 8 years and CHADS2 VASc score was 4.4 ± 1.4. There were 3 DRTs (2 within 6 months, and 1 at 12 months), 4 Type 3A BARC bleeds, and 2 non-cardiac deaths. Patients with DRT had higher baseline thrombin-induced platelet-fibrin clot strength (68.0 ± 1.8 vs. 62.7 ± 4.7 mm, p = 0.06); FCS (35.6 ± 6.0 vs. 24.4 ± 6.6 mm, p = 0.009); and D-dimer (1712 ± 2330 vs. 283 ± 213 ng/mL, p = 0.001). At baseline, 5 patients had all 3 factors associated with high thrombotic risk and 2 experienced a DRT within 6 months. Patients with Type 3A BARC bleeding had lower baseline collagen-induced and 45-day ADP-induced PA (p < 0.01 for both). DRT following LAAC was associated with a baseline prothrombogenic profile whereas bleeding was associated with low platelet reactivity. These preliminary findings warrant further validation and have future implications on patient selection and adjunctive antithrombotic therapy following LAAC.Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT03040622 .

TEG®6s system measures the contributions of both platelet count and platelet function to clot formation at the site-of-care.

Knowledge of platelet count and function is key to ensuring appropriate hemostatic management. We hypothesized that the novel, portable TEG®6s coagulation assessment system could evaluate the contribution of both platelet count and function to clot formation. Whole-blood samples with variable platelet counts were prepared from healthy volunteers. Platelet function was adjusted using seven concentrations of abciximab and evaluated by light transmission aggregometry (LTA) with TRAP agonist. Maximum amplitude (MA), reaction time (R) and activated clotting time (ACT) were assessed in citrated kaolin (CK), CK with heparinase (CKH), citrated RapidTEG® (CRT), and citrated functional fibrinogen (CFF) assays. Positive correlations were observed between platelet count and CK.MA, CKH.MA, and CRT.MA (p < .0001), and CK.R, CKH.R, and CRT.ACT (p < .05). Platelet count could be accurately quantified in the range 28-91 k/µL, 28-86 k/µL and 28-74 k/µL for CK.MA, CKH.MA, and CRT.MA, respectively. CK.MA, CKH.MA, and CRT.MA showed significant negative relationships with abciximab concentration (p < .001). Platelet function inhibition was detected by all three assays at >68% measured by LTA and quantified in the range 68.4-82% (CK), 69.4-88% (CKH), and 69.7-76% (CRT). This demonstrates the TEG®6s analyzer can accurately evaluate platelet count and function at the site-of-care.

A randomized comparison of manual pressure versus figure-of-eight suture for hemostasis after cryoballoon ablation for atrial fibrillation.

INTRODUCTION: Cryoballoon ablation is commonly used to treat atrial fibrillation (AF). Femoral vein hemostasis after cryoballoon ablation for AF is routinely achieved with manual pressure (MP) after reversal of heparin and reassessment of the activated clotting time, or with a figure-of-eight suture (F8). The purpose of this randomized trial was to compare these two techniques for femoral vein hemostasis after cryoballoon ablation for AF in a patient population predominantly on novel anticoagulants (NOAC). METHODS AND RESULTS: Seventy consecutive patients who underwent cryoballoon ablation were randomized to either the MP or F8 for femoral vein hemostasis. Clinical and procedural characteristics were similar between the groups with the majority of patients treated with a NOAC. The total time in the electrophysiology laboratory for the MP group and the F8 group (197 ± 37 minutes vs 167 ± 36 minutes, respectively; P = .02), and the time from sheath removal until the patient left the laboratory (28 ± 9 minutes vs 20 ± 5 minutes, respectively; P < .0001) were significantly less in the F8 group. Additional pressure for hemostasis in the recovery suite was required more often in the MP Group, as opposed to the F8 group (29% vs 3%; P = .003). No major bleeding occurred and the rate of minor hematomas was statistically similar. CONCLUSIONS: Hemostasis obtained with a F8 suture after cryoballoon ablation for AF is associated with significantly less patient time in the electrophysiology laboratory, and an improved safety profile, compared with manual hemostasis, even amongst patients treated with a NOAC.

Noncanonical Matrix Metalloprotease 1-Protease-Activated Receptor 1 Signaling Drives Progression of Atherosclerosis.

OBJECTIVE: Protease-activated receptor-1 (PAR1) is classically activated by thrombin and is critical in controlling the balance of hemostasis and thrombosis. More recently, it has been shown that noncanonical activation of PAR1 by matrix metalloprotease-1 (MMP1) contributes to arterial thrombosis. However, the role of PAR1 in long-term development of atherosclerosis is unknown, regardless of the protease agonist. APPROACH AND RESULTS: We found that plasma MMP1 was significantly correlated (R=0.33; P=0.0015) with coronary atherosclerotic burden as determined by angiography in 91 patients with coronary artery disease and acute coronary syndrome undergoing cardiac catheterization or percutaneous coronary intervention. A cell-penetrating PAR1 pepducin, PZ-128, currently being tested as an antithrombotic agent in the acute setting in the TRIP-PCI study (Thrombin Receptor Inhibitory Pepducin-Percutaneous Coronary Intervention), caused a significant decrease in total atherosclerotic burden by 58% to 70% (P<0.05) and reduced plaque macrophage content by 54% (P<0.05) in apolipoprotein E-deficient mice. An MMP1 inhibitor gave similar beneficial effects, in contrast to the thrombin inhibitor bivalirudin that gave no improvement on atherosclerosis end points. Mechanistic studies revealed that inflammatory signaling mediated by MMP1-PAR1 plays a critical role in amplifying tumor necrosis factor α signaling in endothelial cells. CONCLUSIONS: These data suggest that targeting the MMP1-PAR1 system may be effective in tamping down chronic inflammatory signaling in plaques and halting the progression of atherosclerosis.

Gender differences in thrombogenicity among patients with angina and non-obstructive coronary artery disease.

Women more often present with angina and non-obstructive coronary artery disease (ANOCA) and have poorer clinical outcomes than men. These findings may be related to sex associated differences in inflammation and thrombogenicity. Consecutive patients (n = 134) with ANOCA (luminal diameter stenosis < 50%) undergoing elective cardiac catheterization were included in post hoc analysis of Multi-Analyte, thrombogenic, and Genetic Markers of Atherosclerosis (MAGMA, NCT01276678) study. Patients with prior revascularization, coronary artery bypass grafting or myocardial infarction were excluded. Blood for thromboelastography, oxidized LDL ß2-glycoprotein complex (AtherOx), oxidized-LDL, lipid profile, and urine for 11-dehydrothromboxane B2 (dTxB2) were obtained before catheterization. All women (n = 75) were post-menopausal and tended to be older than men (61.4 ± 10.6 vs. 58.6 ± 9.9 year, p = 0.12), and were significantly more thrombogenic with higher thrombin-induced platelet-fibrin strength (TIP-FCS, mm) (68.0 ± 4.5 vs. 64.5 ± 6.2 mm, p = 0.001), clotting index (0.35 ± 2.22 vs. - 0.72 ± 2.75, p = 0.02), K (measure of the speed to reach 20 mm of clot strength from an amplitude of 2 mm) (2.2 ± 1.6 vs. 1.7 ± 0.5 min, p = 0.01), and fibrinogen activity (degrees) (66.6 ± 7.1 vs. 62.9 ± 7.5, p = 0.009). Markers of inflammation were not significantly different between the two groups. Women had higher total cholesterol, total LDL, LDL subtypes 1 and 2, total HDL, HDL subtypes 2 and 3, and ApoA1 (p < 0.05 for all). On multivariate regression, TIP-FCS remained significantly higher in women (p < 0.0001). Women with ANOCA are more thrombogenic than men. This fundamental difference in thrombogenicity may affect gender-related outcomes and warrants further investigation.

"Blueprinting" thrombogenicity and antithrombotic drug response at the bedside in patients presenting emergently with symptoms of acute stroke.

Early assessment of thrombogenicity and antithrombotic drug effects may be important for therapeutic decision making in patients presenting with acute stroke. In this prospective, single center, pilot study, a bedside thrombelastography assay (TEG6s) was used to measure thrombogenicity and antithrombotic drug response in serial patients presenting emergently with symptoms of acute stroke (n = 90). TEG6s measures were compared against diagnosis obtained by NIH Stroke Scale/Score and imaging. Acute ischemic stroke (AIS) was diagnosed in 30 patients, intracerebral hemorrhage (ICH) in 19, transient ischemic attack (TIA) in 10 and stroke mimic (SM) in 31. Patients with AIS had a higher prevalence of A-Fib (33.3% vs. 11.6%, p = 0.01), and ACE inhibitor use (56.3% vs. 21.6%, p < 0.001) compared to combined non-AIS group. Time to initial clot formation (R) was shorter in AIS vs. TIA, ICH, and SM (p < 0.05). Comparing patients with AIS and combined non-AIS group the AUC for R was 0.83 (cut point of ≤ 4.8, sensitivity = 67%; specificity = 84%, p < 0.001). In AIS patients, 46% had suboptimal response (< 30% MAAA inhibition) to aspirin and 80% of patients on P2Y12 therapy had high platelet reactivity (> 50% ADP-induced platelet aggregation). Patients receiving tissue plasminogen activator had significant reduction in clot strength and near complete lysis at 30 min which normalized within 2 h after treatment (p < 0.001 for both). The rapid bedside measurement of thrombogenicity and antithrombotic drug effects is feasible in patients presenting with symptoms of acute stroke. Our preliminary data suggest that AIS is associated with faster ex-vivo clot formation, and poor antiplatelet response. Future study of the TEG6s to "blueprint" hemostasis is warranted in the stroke population.

Genome-wide and candidate gene approaches of clopidogrel efficacy using pharmacodynamic and clinical end points-Rationale and design of the International Clopidogrel Pharmacogenomics Consortium (ICPC).

RATIONALE: The P2Y12 receptor inhibitor clopidogrel is widely used in patients with acute coronary syndrome, percutaneous coronary intervention, or ischemic stroke. Platelet inhibition by clopidogrel shows wide interpatient variability, and high on-treatment platelet reactivity is a risk factor for atherothrombotic events, particularly in high-risk populations. CYP2C19 polymorphism plays an important role in this variability, but heritability estimates suggest that additional genetic variants remain unidentified. The aim of the International Clopidogrel Pharmacogenomics Consortium (ICPC) is to identify genetic determinants of clopidogrel pharmacodynamics and clinical response. STUDY DESIGN: Based on the data published on www.clinicaltrials.gov, clopidogrel intervention studies containing genetic and platelet function data were identified for participation. Lead investigators were invited to share DNA samples, platelet function test results, patient characteristics, and cardiovascular outcomes to perform candidate gene and genome-wide studies. RESULTS: In total, 17 study sites from 13 countries participate in the ICPC, contributing individual patient data from 8,829 patients. Available adenosine diphosphate-stimulated platelet function tests included vasodilator-stimulated phosphoprotein assay, light transmittance aggregometry, and the VerifyNow P2Y12 assay. A proof-of-principle analysis based on genotype data provided by each group showed a strong and consistent association between CYP2C19*2 and platelet reactivity (P value=5.1 × 10-40). CONCLUSION: The ICPC aims to identify new loci influencing clopidogrel efficacy by using state-of-the-art genetic approaches in a large cohort of clopidogrel-treated patients to better understand the genetic basis of on-treatment response variability.