Plasminogen Activator Inhibitor Type 1: A Possible Novel Biomarker of Late Pituitary Dysfunction after Mild Traumatic Brain Injury.

More than 80% of traumatic brain injury (TBI) patients suffer from mild TBI (mTBI). However, even mTBI carries the risk of late pituitary dysfunction. A predictive biomarker at the time of injury that could identify patients who subsequently may develop permanent pituitary dysfunction would help to direct patients toward endocrine care. We enrolled 508 TBI patients (406 with mTBI) into our study. Blood samples were collected for identification of predictive biomarkers of late pituitary dysfunction at the time of admission. Follow-up blood samples were collected between 6 and 12 months after the TBI and were evaluated for pituitary function. Of the 406 mTBI patients, 76 were available for follow-up. Pre-existing mild pituitary dysfunction was found for 15 patients based on hormone levels at the time of injury. Of the remaining 61 patients, 10 have shown deficiency in at least one pituitary hormone: 4 had growth hormone deficiency, 3 gonadotropin, 2 thyrotropin, and 1 patient combined gonadotropin and thyrotropin deficiency. Hence, newly developed pituitary hormone deficiency was found in 16% of mTBI patients. Neither the cause of mTBI nor its complications were predictive of late pituitary dysfunction. Of the hemostasis parameters studied, lower plasminogen activator inhibitor type 1 (PAI-1) level at the time of injury was found to be predictive for the development of late pituitary dysfunction; sensitivity, specificity, and positive and negative predictive values were 80%, 67%, 32%, and 94%, respectively. Even mTBI carries a substantial risk of endocrine consequences. Serum PAI-1 level at the time of TBI may serve as a predictive biomarker of late pituitary dysfunction in mTBI patients.

Low thrombin generation predicts poor prognosis in ischemic stroke patients after thrombolysis.

Thrombolysis by intravenous recombinant tissue plasminogen activator (rt-PA) is an effective therapy in acute ischemic stroke (AIS). Thrombin generation test (TGT) is a global hemostasis test providing information about the speed and amount of generated thrombin in plasma. Here we aimed to find out whether results of this test before the initiation of thrombolysis might predict outcomes. Study population included 120 consecutive AIS patients, all within 4.5 hours of their symptom onset, who underwent thrombolysis by rt-PA. Blood samples were collected from all patients upon admission and TGT was performed using platelet poor plasma. Clinical data of patients including the NIHSS were registered at admission, day 1 and 7 after therapy. The ASPECT score was assessed using CT images taken before and 24 hours after thrombolysis. Long-term functional outcome was defined 3 months after the event by the modified Rankin Scale. Endogenous Thrombin Potential (ETP) and Peak Thrombin were significantly lower in patients with cardioembolic IS. Symptomatic intracranial hemorrhage (SICH) was found in 6 patients and was significantly associated with low ETP and Peak Thrombin levels. A multiple logistic regression model revealed that an ETP result in the lower quartile is an independent predictor of mortality within the first two weeks (OR: 6.03; 95%CI: 1.2-30.16, p<0.05) and three months after the event (OR: 5.28; 95%CI: 1.27-21.86, p<0.05). Low levels of ETP and Peak Thrombin parameters increase the risk of therapy associated SICH. A low ETP result is an independent predictor of short- and long-term mortality following thrombolysis.

The Phosphatase Inhibitor Calyculin-A Impairs Clot Retraction, Platelet Activation, and Thrombin Generation.

The aim of this study was to investigate the effect of the serine/threonine protein phosphatase inhibitor, calyculin-A (CLA), on clot formation and on the procoagulant activity of human platelets. Platelet-rich plasma (PRP) samples were preincubated with buffer or CLA and subsequently platelets were activated by the protease-activated receptor 1 (PAR-1) activator, thrombin receptor activating peptide (TRAP). Clot retraction was detected by observing clot morphology up to 1 hour, phosphatidylserine- (PS-) expression was studied by flow cytometry, and thrombin generation was measured by a fluorimetric assay. For the intracellular Ca2+ assay, platelets were loaded with calcium-indicator dyes and the measurements were carried out using a ratiometric method with real-time confocal microscopy. CLA preincubation inhibited clot retraction, PS-expression, and thrombin formation. TRAP activation elicited Ca2+ response and PS-expression in a subset of platelets. The activated PRP displayed significantly faster and enhanced thrombin generation compared to nonactivated samples. CLA pretreatment abrogated PS-exposure and clot retraction also in TRAP-activated samples. As a consequence of the inhibitory effect on calcium elevation and PS-expression, CLA significantly downregulated thrombin generation in PRP. Our results show that CLA pretreatment may be a useful tool to investigate platelet activation mechanisms that contribute to clot formation and thrombin generation.

Laboratory characterization of leukemic cell procoagulants.

BACKGROUND: In acute myeloid leukemias, there is an increased chance to develop thrombotic disorders. We hypothesized that in addition to leukemic promyelocytes, monocytic leukemia cells may also have a higher procoagulant activity. METHODS: Fibrin formation was assessed by a one-stage clotting assay using a magnetic coagulometer. The thrombin generation test (TGT) of magnetically isolated normal human monocytes, intact leukemic cells and their isolated microparticles was performed by a fluorimetric assay. Phosphatidylserine (PS) expression of leukemic cells and microparticle number determinations were carried out by flow cytometry. RESULTS: All cell lines displayed a significant procoagulant potential compared to isolated normal human monocytes. In the TGT test, the mean of lagtime and the time to peak parameters were significantly shorter in leukemic cells (3.9-4.7 and 9.9-10.3 min) compared to monocytes (14.9 and 26.5 min). The mean of peak thrombin in various monocytic leukemia cell lines was 112.1-132.9 nM vs. 75.1 nM in monocytes; however, no significant difference was observed in the ETP parameter. Factor VII-deficient plasma abolished all procoagulant activity, whereas factor XII-deficient plasma did not affect the speed of fibrin formation and thrombin generation but modulated the amount of thrombin. Factor XI-deficient plasma affected the time to peak values in one leukemic cell line and also attenuated peak thrombin. Leukemia cell-derived microparticles from all three cell lines exerted a procoagulant effect by significantly shortening the lagtime in TGT; there was a nonsignificant difference in case of ETP parameter. CONCLUSIONS: All investigated monocytic leukemia cell lines exhibited significant thrombin generation. This phenomenon was achieved by the procoagulants on the surface of leukemic cells as well as by their microparticles.

Haemodiafiltration elicits less platelet activation compared to haemodialysis.

BACKGROUND: Mortality in patients with end-stage renal disorders is often a consequence of cardiovascular complications. Renal replacement therapies may contribute to this morbidity by promoting cellular activation. In renal failure patients peripheral blood samples were investigated for platelet and endothelial cell activation markers to compare the effects of haemodiafiltration (HDF) and haemodialysis (HD). METHODS: Overall 28 patients were included in the study. Platelet P-selectin and leukocyte - platelet heterotypic aggregates were studied by flow cytometry. Soluble P- and E-selectin values were determined by ELISA, while von Willebrand factor (vWF) antigen levels were measured by immunoturbidimetry. Statistical analysis was done by the SPSS v22 software. RESULTS: Platelet surface P-selectin was below 3.0 % in healthy controls, but it was higher during the dialysis after 4 h, 8 % and 14.3 % in HDF and HD, respectively. Monocyte-platelet heterotypic aggregates were significantly elevated after 4 h in both treatments, up to 69.2 % in HDF and to 82.9 % in HD. Soluble P-selectin levels were also significantly elevated by the end of both treatment procedures (p < 0.001), vWF antigen values, however, showed elevation only during HD treatment. CONCLUSIONS: The attenuated platelet activating effects of HDF compared to HD may contribute to a less unfavourable vascular effect in this treatment modality.

Laboratory evaluation of a flow cytometric BCR-ABL immunobead assay.

BACKGROUND: A new flow cytometric (FC) BCR-ABL immunobead assay has been developed recently. Here we present the laboratory evaluation of the commercially available kit. METHODS: Mononuclear cells were isolated, lysed and processed according to the instructions of the manufacturer. Anti-BCR antibodies adsorbed to capture beads bind the BCR-ABL fusion proteins of the lysed cells, a phycoerythrin (PE)-conjugated anti-ABL antibody is the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Detection of t(9;22)(q34;q11) translocation was carried out with a quantitative PCR assay. RESULTS: MFI results of 20 normal peripheral blood samples were 88±8 (mean±SD), CV 9%. K562 cells were used as positive control. Within-batch imprecision was excellent (3.7% in the normal and 10% in the pathological range). Cut-off was chosen at MFI 112, where both sensitivity and specificity were 100%. Altogether 17 chronic myeloid leukemia (CML) and 16 acute leukemia samples were analyzed. All PCR positive samples (n=14) were positive with the FC method and negative results were also concordant (n=15). Frozen cell lysates can be stored up to 4 weeks without significant decrease of MFI signal. CONCLUSIONS: The FC BCR-ABL assay is a fast, reproducible and reliable method that may be incorporated into standard flow cytometric protocols to help clinical decision-making.