Ann M. Cullinane

The effect of red blood cells on thrombin generation

We have developed an assay of thrombin generation in clotting whole blood. Because our goal was to study patients with red blood cell diseases that are associated with thrombosis, the initial evaluation of this assay analysed the effect of the haematocrit and haemolysate on the total amount of thrombin activity generated and the maximal concentration of thrombin achieved. Both of these parameters were proportional to the haematocrit throughout a wide range of clinically relevant cell concentrations. Red cell lysate also augmented thrombin generation. Because this effect was removed by filtration of the lysate, we propose that it was related to red cell microparticles.

Incidence and risk factors of bleeding-related adverse events in patients with chronic lymphocytic leukemia treated with ibrutinib

Ibrutinib is associated with bleeding-related adverse events of grade ≤2 in severity, and infrequently with grade ≥3 events. To investigate the mechanisms of bleeding and identify patients at risk, we prospectively assessed platelet function and coagulation factors in our investigator-initiated trial of single-agent ibrutinib for chronic lymphocytic leukemia. At a median follow-up of 24 months we recorded grade ≤2 bleeding-related adverse events in 55% of 85 patients. No grade ≥3 events occurred. Median time to event was 49 days. The cumulative incidence of an event plateaued by 6 months, suggesting that the risk of bleeding decreases with continued therapy. At baseline, von Willebrand factor and factor VIII levels were often high and normalized on treatment. Platelet function measured via the platelet function analyzer (PFA-100™) was impaired in 22 patients at baseline and in an additional 19 patients on ibrutinib (often transiently). Collagen and adenosine diphosphate induced platelet aggregation was tested using whole blood aggregometry. Compared to normal controls, response to both agonists was decreased in all patients with chronic lymphocytic leukemia, whether on ibrutinib or not. Compared to untreated chronic lymphocytic leukemia patients, response to collagen showed a mild further decrement on ibrutinib, while response to adenosine diphosphate improved. All parameters associated with a significantly increased risk of bleeding-related events were present at baseline, including prolonged epinephrine closure time (HR 2.74, P=0.012), lower levels of von Willebrand factor activity (HR 2.73, P=0.009) and factor VIII (HR 3.73, P=0.0004). In conclusion, both disease and treatment-related factors influence the risk of bleeding. Patients at greater risk for bleeding of grade ≤2 can be identified by clinical laboratory tests and counseled to avoid aspirin, non-steroidal anti-inflammatory drugs and fish oils. ClinicalTrials.gov identifier NCT01500733

Serologic evidence of heparin sensitization in cancer patients receiving heparin flushes of venous access devices.

Abstract Cancer patients with venous access devices (VADs) often receive daily flushes of heparin. Even this relatively small heparin exposure has been reported to induce immune-mediated thrombocytopenia. To estimate how frequently this occurs we tested for heparin-related antibodies in 49 patients receiving daily heparin flushes of their VADs. Although one-third of the patients showed evidence of heparin sensitization on at least one occasion during their surveillance, their antibody titers were generally low and typical of those found in other cohorts of patients who become sensitized to heparin but do not develop secondary thrombocytopenia. However, one patient developed a positive serotonin release assay indicative of a more significant sensitization, but without thrombocytopenia. Therefore, our observations suggest that heparin-induced thrombocytopenia (HIT) related to heparin flushes of VADs is uncommon but still an important diagnosis to entertain.

Inflammation, TNFα, and endothelial dysfunction link lenalidomide to venous thrombosis in chronic lymphocytic leukemia

Patients receiving lenalidomide are at an increased risk for deep venous thrombosis (DVT). Here, we prospectively investigated the DVT risk in patients with relapsed chronic lymphocytic leukemia (CLL) treated with lenalidomide (n = 32). Five patients developed six incidents of DVT over 1 year for an annual incidence of 16%. Three of these were considered drug‐related. Median time to DVT was 105 days (range 56–259 days). No pulmonary embolism was detected. Hypercoagulability screen before study entry was negative in all patients who subsequently developed DVTs. Compared to normal volunteers CLL patients had increased baseline levels of D‐dimer, thrombin‐antithrombin, soluble vascular endothelial adhesion molecule 1 (sVCAM‐1), and thrombomodulin (p < 0.001). After 1 week on lenalidomide D‐dimer, thrombomodulin, sVCAM‐1, factor VIII, TNFα, and C‐reactive protein were significantly increased while protein C was decreased (p < 0.001). In patients with lenalidomide‐related DVTs, TNFα, and sVCAM‐1 were more strongly upregulated than in all other patients (p < 0.05) and TNFα and sVCAM‐1 levels were significantly correlated (r = 0.65, p < 0.001). These data link lenalidomide associated DVTs with TNFα upregulation and endothelial cell dysfunction and suggest that aspirin may have a role for DVT prophylaxis in these patients. Am. J. Hematol., 2011.

Comparison of the effect of histidine-rich glycoprotein and 6-aminohexanoic acid on plasmin production and fibrinolysis in vitro.

Because histidine-rich glycoprotein binds to the kringle 1-3 domain of plasminogen, it may affect fibrinolysis by reducing fibrin-dependent plasmin production, and in this way it could be mechanistically analogous to 6-aminohexanoic acid. We tested this hypothesis by comparing the effects of histidine-rich glycoprotein and 6-aminohexanoic acid in an in vitro assay of fibrin-dependent plasmin production mediated by tissue plasminogen activator. Whereas 1 mM of 6-aminohexanoic acid increased the K(m) of the reaction from approximately 0.22 microM to approximately 1.7 microM, 2 microM of histidine-rich glycoprotein had no discernible effect. Similar results were obtained in an assay based upon fibrin clot lysis. Therefore, we could not document an effect of histidine-rich glycoprotein on the rate of fibrin-dependent plasmin production.

Biochemical dynamics relevant to the safety of low-dose, intraclot alteplase for deep vein thrombosis

Intraclot tissue plasminogen activator (tPA) has been shown to be an effective treatment for deep vein thrombosis (DVT) (Radiology 2008;246:619 and J Vasc Interv Radiol 2011;22:1107). We sought to correlate pharmacokinetics of tPA, fibrinogen, fibrinolytic inhibitors, and D-dimers with the safety and efficacy of intraclot tPA. Thirty subjects received intraclot tPA for lower extremity DVT by infiltrating the thrombus with ≤10 mg doses tPA in an open-label study, using a pulse-spray catheter. We measured various parameters over 8 h following a first dose of tPA. Mean tPA levels of 75 units per mL (95% confidence interval 19-131 units/mL) were seen immediately after administration of a mean tPA dose of 8.0 mg (SD 1.5 mg). tPA levels returned to baseline within 2 h of completion of treatment. Plasminogen activator inhibitor-1 (PAI-1) was consumed following tPA treatment, but rose to levels significantly greater than baseline (P < 0.001). Fibrinogen decreased slightly, but remained >125 mg/dL for all subjects. α2-antiplasmin decreased from a mean of 115 units/mL to 56 units/mL after tPA administration (P < 0.001) and remained decreased for 8 h. Plasminogen at baseline (112 units/mL) decreased to 89 units/mL immediately after tPA administration (P < 0.001) and was unchanged thereafter. D-dimer levels were >20 μg/mL in all but 4 subjects, one of whom was the only one to fail to achieve clot lysis. The safety of low-dose, intraclot tPA is due to its short persistence in the circulation, lack of hypofibrinogenemia, and a reflexive rise of PAI-1. Subjects whose D-dimers remain <20 μg/mL are at risk of not achieving thrombolysis.

In vitro characterization of a monoclonal IgGκ from a patient with planar xanthomatosis

Objective:  To characterize a monoclonal IgGκ (MAb) from a patient with planar xanthoma that precipitated with serum lipids at reduced temperature.

Parameters of coagulant and fibrinolytic capacity and activity in postmenopausal women: within-subject variability.

We have analyzed the within-subject variability of a battery of parameters of coagulant and fibrinolytic capacity and activity in postmenopausal women. We observed large differences in within-subject variability among the tests and have demonstrated how such data can be used to estimate the number of times a parameter must be measured to produce a statistically adequate sample.