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Increasing D-dimer (DD) levels after discontinuation of vitamin K antagonist (VKA) therapy indicate an increased risk of recurrence of venous thromboembolism (VTE). However, after discontinuation of d irect-acting n on-VKA o ral a nti c oagulants (DOACs or NOACs) the extent of coagulation activation and its clinical impact is unknown. Blood samples were collected from consenting patients with proximal VTE at the end of anticoagulation treatment with apixaban (n = 37), dabigatran (n = 17), rivaroxaban (n = 9) or VKA (n = 184) and 4 weeks later. DD, prothrombin fragments F1 + 2 (F1 + 2) and thrombin–antithrombin complexes (TAT) were measured. All patients underwent follow-up at 12 months to establish recurrent VTE or death from any cause. Irrespective of the treatment, DD and F1 + 2 but not TAT demonstrated a similar increase between baseline and week 4. At 12 months, 18 patients (7.3%) had recurrent VTE and two (0.8%) had died. For all patients and subgroups of VKA and DOAC, positive likelihood ratios were numerically higher for baseline values but only TAT values at 4 weeks were found to be related to a small increase of outcome event likelihood (2.6; 95%CI 1.23-5.50), which was driven by VKA patients (3.1; 95%CI 1.32-7.30) and not by DOAC patients (2.27; 95%CI 0.52-9.95). For all parameters, negative likelihood ratios were not predictive. In logistic regression analysis, only ΔTAT (optimal cut-off > 178% from baseline demonstrated a significant risk increase for VTE/death (odds ratio 3.76; 95% confidence interval 1.46-9.68; p = 0.006). In conclusion, the concept of testing coagulation activation parameters may also be transferred to VTE patients at the end of DOAC therapy. For patients with an increase of TAT levels within 4 weeks after treatment discontinuation (> 178% from baseline) is associated with an increased risk for VTE recurrence or death at 12 months.

Aims Variability in responsiveness to clopidogrel is a clinical problem in secondary prevention after cerebral ischaemia which has been suggested to be linked to competitive metabolization of clopidogrel and cytochrome P450 (CYP) 3A4-oxidated statins such as simvastatin. We assessed the hypothesis that simvastatin, in contrast to CYP 2C9-metabolized fluvastatin, reduces clopidogrel-mediated platelet inhibition. Methods We performed a randomized, double-blind, double-dummy, two period crossover study in 13 patients with cerebral ischaemia (8F, 5 M), aged 64.1 ± 8.0 years (mean ± SD). After a 14 day period in which all patients received 75 mg clopidogrel day−1, patients additionally received either 20 mg simvastatin day−1 or 80 mg fluvastatin day−1 for 14 days. Regimens were crossed over after a 14 day wash-out period and switched regimens were continued for another 14 days. Platelet aggregation, clopidogrel active metabolite (CAM) plasma concentrations and routine laboratory parameters including prothrombin time (PT) Quick percent value were assessed at baseline and following each treatment phase. Results Clopidogrel reduced platelet aggregation in all patients as expected. Platelet aggregation and CAM plasma concentrations were unaltered when simvastatin or fluvastatin was added to clopidogrel. Simvastatin decreased PT Quick percent value (decrease from 109 ± 10.5% to 103 ± 11%, P < 0.05) when combined with clopidogrel but there was no such change following treatment with fluvastatin and clopidogrel. Conclusions Our data indicate that treatment with CYP 3A4-metabolized simvastatin does not jeopardize clopidogrel-mediated inhibition of platelet aggregation. After co-administration of simvastatin and clopidogrel we observed a decrease in the PT Quick percent value which could be due to simvastatin-induced reduction of activity of prothrombin fragment 1 + 2.

Rotational thromboelastometry (ROTEM), a method for the clinical characterisation of blood clot formation and fibrinolysis, has been applied to study the acid-induced gelation of cross-linked casein. The results were compared to those from small amplitude oscillatory shear (SAOS) experiments that were carried out on the same samples: acid casein in phosphate buffer and reconstituted skim milk were treated with microbial transglutaminase to achieve cross-linking of casein and to obtain gels with a broad range of stiffness upon acidification with glucono-δ-lactone. Gelation onset was detected consistently 2 min later by ROTEM compared to SAOS, which my be attributed to the differences in the onset criteria of both methods. Comparison of maximum clot firmness (ROTEM) and maximum storage modulus (rheometry) as indicators for gel stiffness revealed a strong non-linear relationship, that was successfully fitted to the common model of ROTEM clot elasticity through adding a proportionality coefficient. From the results of this study it can be concluded that ROTEM might serve as a useful tool for characterising the acid-induced gelation of proteins that are available in only small quantities.

Aspirin-like defect (ALD) is a rare, mostly autosomal dominant inherited dysfunction of the intraplatelet arachidonic acid (AA) pathway leading to impaired thromboxane A2 signalling. We aimed to establish diagnostic criteria for ALD diagnosis and present clinical and laboratory phenotypes of 52 individuals from 17 unrelated families. Platelet in vitro function was determined on the basis of platelet aggregation response (PAR) to AA, adenosine diphosphate, collagen and ristocetin as well as PFA-100 closure times (CT). Using impaired PAR to AA (< or =10%) as the mandatory diagnostic criterion, ALD could be confirmed in 17 patients. Subsequently, family members were investigated and among 35 individuals an additional 13 ALD patients as well as 4 individuals with mild ALD (PAR to AA: 19-32%) were identified. At least one bleeding symptom was reported by 25 (74%) ALD patients and prolonged CT was detected in 24 (71%) of the cases, both significantly correlated with impaired PAR to AA (P = 0.001 and P = 0.002, respectively). An estimated 0.6% prevalence was determined for ALD in our paediatric patients with suspected coagulation disorders. Due to the mild bleeding symptoms, ALD is probably underdiagnosed. If ALD is suspected, PAR to AA is suitable for the identification of individuals at risk of increased haemorrhage.

Acute ischemic stroke (AIS) patients receiving non-vitamin-K antagonist oral anticoagulants (NOAC) are commonly excluded from thrombolytic therapy, as interpretation of coagulation tests remains unclear. We aimed to investigate the applicability of a novel institutional protocol for thrombolysis based on current expert recommendations and NOAC specific coagulation assessment. We included hospitalized AIS patients receiving NOAC for at least 24 h and consecutive AIS patients not receiving NOAC into a prospective study. We performed standard coagulation tests and specific tests for dabigatran, rivaroxaban and apixaban plasma levels. We studied 65 patients: mean age 72 ± 13 years, 30 (46 %) male, median NIHSS score 3 (IQR 6). Fifteen (23 %) were on NOAC treatment (5 dabigatran, 5 rivaroxaban, and 5 apixaban, respectively) and 50 (77 %) were not. In patients without NOAC, dabigatran was not detectable (0 ng/ml), and plasma levels of rivaroxaban (median: 10.0 ng/ml, IQR 7.0) and apixaban (7.2 ng/ml, IQR 6.7) were below our lower thresholds that allow thrombolysis. In patients with dabigatran pre-treatment, trough levels (58.0 ng/ml, IQR 143.0) were below our upper threshold that would allow thrombolysis in 3/5 patients. In patients receiving rivaroxaban, trough level (68.0 ng/ml, IQR 64.0) was below our predefined upper thresholds that would allow thrombolysis in 4/5 patients. In all patients on apixaban, trough level was above our predefined threshold of 40 ng/ml that precludes thrombolysis (98.2 ng/ml, IQR 84.3). Predefined thresholds of NOAC plasma levels in the decision of thrombolysis in NOAC treated AIS patients might supplement routine coagulation tests and should be validated in a larger study population.

Factor V Leiden is a well-known risk factor for venous thrombosis. The dual role of factor V as a coagulatory and anticoagulatory cofactor permits the assumption that further mutations in the factor V gene are of importance in the study of the risk of thrombosis. Relevant studies to date have given rise to a controversy over this risk for the HR2 haplotype. For the G allele, defined in our work group as a G at the nucleotide positions 2391, 2663, 2684 and 2863, there have been to date no other investigations of thrombotic risk. In a case-control study on 347 patients with deep venous thrombosis (DVT) and 282 controls, we investigated the association of the HR2 haplotype and the G allele with DVT. We found no association between HR2 haplotype and DVT [odds ratio (OR) 0.87; 95% confidence interval (CI) 0.58-1.30; P = 0.537]. The frequency of the G allele was, on the contrary, higher in the control group than among the patients (OR 0.68, 95% CI: 0.53 to 0.89; P = 0.005). The factor V activity of the HR2 carriers was lower than that of the wild type and G allele carriers. The HR2 haplotype exhibited a moderate influence on activated protein C response. This study presented no evidence of thrombotic risk for the HR2 haplotype alone. The results here permit the assumption of a protective effect of the G allele. The source of a possible protective influence of the G allele on thrombotic risk is at present unclear.

Variability in responsiveness to clopidogrel is a clinical problem in secondary prevention after cerebral ischaemia which has been suggested to be linked to competitive metabolization of clopidogrel and cytochrome P450 (CYP) 3A4-oxidated statins such as simvastatin. We assessed the hypothesis that simvastatin, in contrast to CYP 2C9-metabolized fluvastatin, reduces clopidogrel-mediated platelet inhibition.We performed a randomized, double-blind, double-dummy, two period crossover study in 13 patients with cerebral ischaemia (8F, 5 M), aged 64.1 ± 8.0 years (mean ± SD). After a 14 day period in which all patients received 75 mg clopidogrel day(-1) , patients additionally received either 20 mg simvastatin day(-1) or 80 mg fluvastatin day(-1) for 14 days. Regimens were crossed over after a 14 day wash-out period and switched regimens were continued for another 14 days. Platelet aggregation, clopidogrel active metabolite (CAM) plasma concentrations and routine laboratory parameters including prothrombin time (PT) Quick percent value were assessed at baseline and following each treatment phase.Clopidogrel reduced platelet aggregation in all patients as expected. Platelet aggregation and CAM plasma concentrations were unaltered when simvastatin or fluvastatin was added to clopidogrel. Simvastatin decreased PT Quick percent value (decrease from 109 ± 10.5% to 103 ± 11%, P0.05) when combined with clopidogrel but there was no such change following treatment with fluvastatin and clopidogrel.Our data indicate that treatment with CYP 3A4-metabolized simvastatin does not jeopardize clopidogrel-mediated inhibition of platelet aggregation. After co-administration of simvastatin and clopidogrel we observed a decrease in the PT Quick percent value which could be due to simvastatin-induced reduction of activity of prothrombin fragment 1 + 2.

Background: Acute ischemic stroke (AIS) patients receiving novel oral anticoagulants (NOAC) are commonly excluded from thrombolytic therapy as reliable bedside tests for anticoagulation monitoring are lacking. We aimed to prospectively investigate the applicability of our institutional protocol for the management of AIS patients on NOAC that is based on expert recommendations Methods: We studied consecutive AIS patients not receiving NOAC and hospitalized general patients receiving NOAC. We performed standard coagulation tests including INR, activated partial thromboplastin time (aPTT), thrombin time (TT) and trough level tests for dabigatran, rivaroxaban and apixaban. In AIS patients, blood samples were taken immediately after admission. Results: We enrolled 40 patients: mean age 73±15 yrs, 30% male, median NIHSS 3 (range 0-17), 62.5% AIS. Fifteen patients received NOAC (i.e., 5 dabigatran, 5 rivaroxaban, 5 apixaban). In AIS patients not receiving NOAC, coagulation parameters were within normal limits (INR 0.99±0.07, aPTT 27.4±2.6, TT 16.7±1.2) and significantly lower than in those on dabigatran (INR 1.28±0.15, aPTT 39.6±7.4, TT 111.9±40.8; p Conclusions: In AIS patients potentially amenable to thrombolytic therapy, altered routine coagulation tests may point to an intake of NOAC. Predefined values for dabigatran, rivaroxaban and apixaban need to be adjusted in a larger validation study and individually validated by local laboratories to be prepared for emergency situations.

The factor V (FV) B-domain is extremely important to the cofactor function of native FV for activated protein C (APC) in the inactivation of factor VIII (FVIII). In a previous study, we found that in the B-domain coding portion of DNA, the polymorphisms at nucleotide positions 2391, 2663, 2684, and 2863 were associated. In the major allele, all bases are A (A allele) and those in the minor allele are G (G allele). This study concerns itself with the question of whether or not there are differences in the APC response between the A allele and the G allele in plasma samples from persons without the FV Leiden. The APC ratios of homozygous carriers of the major A allele and the minor G allele do not differentiate themselves in classical activated partial thromboplastin time-based assays. In contrast, a test based on the deactivation of FVIII in the tenase complex in homozygous carriers of the minor G allele showed significantly lower APC ratios (P = 0.001) in comparison with the major A allele. The results of the investigation after modification of the test indicate that mutative changes in the B-domain apparently influence the interaction among phospholipids, APC, FV, and protein S. An increase in FVIII through the introduction of the FVIII concentrate Kogenate to the plasma samples was associated with a drop in the APC ratios of both genotypes. After defining 59 age- and sex-based matched pairs without the FV Leiden, the observed frequency of the minor G allele was higher in the non-thrombotic group (33.0%) than in the thrombotic group (22.8%). However, the difference did not reach the level of significance (odds ratio, 0.53; 95% confidence interval, 0.26-1.12). It does, nevertheless, appear possible that a homozygous condition for the minor allele in combination with a defect known to be associated with thrombophilia represents an additional thrombogenetic risk factor.

Hepatic lipase is an enzyme which hydrolyzes triglycerides from plasma lipoproteins and thus takes part in the metabolism of intermediate density lipoproteins and high-density lipoproteins. The search described here concentrated on mutations of the HL gene in 129 patients with combined hypertriglyceridemia/hyperalphalipoproteinemia and in 184 members of 19 families with familial combined hyperlipidemia. Controls were 100 subjects with favorable lipid values (age 46-51 years). Mutation screening and analysis were performed by temperature-gradient gel electrophoresis, allele-specific restriction genotyping, and sequencing. Six different missense mutations and four different silent mutations were found in the HL gene. The alleles Phe-267 and Gln-343 were detected only once in the patient group with hypertriglyceridemia and hyperalphalipoproteinemia and were not detected in the control group. The allele Met-383 was rare in both patients and controls. We found 9.3% of the patients and only 3.0% of controls to be carrying the Val-73-Met missense mutation. The allele Phe-334 was found in 5.43% of patients and in 2.0% of controls. The difference between the frequencies of these alleles was significant between male patients and male controls (Met-73 P=0.044; Phe-334 P=0.047). Also, the summarized odds ratio of 3.28 (95% confidence interval 1.23-8.73) demonstrates that mutation carriers are significantly more prevalent in the patients. Fifteen carriers of the Met-73 allele were found in six families of the familial combined hyperlipidemia group. Furthermore, six carriers of the Phe-334 allele were found in three families of the same group. In comparison to the controls the summarized odds ratio of 2.45 (95% confidence interval 0.89-6.71) barely missed the level of significance. The linkage between genotype and phenotype was incomplete. These results show an association of the missense mutations Val-73-Met and Leu-334-Phe as susceptibility alleles for combined forms of hyperlipidemia.

Arthrocentesis is an essential emergency step in managing patients with acute arthritis. To identify a bacterial infection, Gram staining is performed promptly. However, crystal analysis may not be immediately performed in many facilities. Being considered not to be stable over time, synovial fluid (SF) is sometimes discarded instead of being stored for crystal identification.The aim of this study was to assess the detectability of monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals in SF over a period of 3 days.Consecutive SF samples from 75 joints were analyzed for MSU, CPP crystals, and pH. Two independent observers evaluated the samples by regular light and polarization microscopy immediately after arthrocentesis and after 1, 2, and 3 days at room temperature or at 4°C.Of 75 samples, 27 contained crystals (16 MSU, 6 CPP, 5 both); semiquantitative counts of both MSU and CPP crystals did not change significantly after 3 days. There was no new formation of crystals in any of the crystal-negative samples, which was independent of the storage temperature. Synovial fluid pH was not predictive of crystals and did not change over time.Although immediate workup for microbiology, including Gram stain and culture, is indispensable and well established, crystal analysis may at times not be immediately performed. Our study suggests that when crystal identification cannot be done immediately, it can be safely performed up to 3 days after arthrocentesis when SF is stored at 4°C or even at stable room temperature (20°C).

Rivaroxaban is a direct factor Xa inhibitor, which is rapidly absorbed in the upper gastrointestinal (GI) tract. In large trials, it has been shown to be effective and safe in VTE treatment. However, in these trials patients with morbid obesity were not reported and it is unknown if the standard dosage of 20 mg rivaroxaban is sufficient for bariatric patients, especially after bariatric surgery, which may impact the resorption of rivaroxaban. We report the case of a bariatric patient with high venous thromboembolism risk and instable INR after recent bariatric surgery, who was switched from Vitamin-K antagonists to rivaroxaban. After intake of 20 mg rivaroxaban, plasma concentration were repeatedly measured until 3 h after the second dose using a commercially available chromogenic aXa-assay. Furthermore, INR and aPTT were measured. Peak concentrations of 224.22 ng/ml were observed. After 6 h, plasma concentration decreased to 86.9 ng/ml and remained stable until 12 h (86.32 ng/ml). After 24 h, a trough level of 35.54 ng/ml was observed. The patients INR did immediately increase and remained significantly elevated throughout the day with a slow decrease. Since peak values of rivaroxaban plasma concentrations were in the expected range of published data, we conclude that resorption of rivaroxaban was immediate and not significantly impaired by bariatric surgery of the upper GI tract. Consequently, no dose adjustments seem to be necessary in this high-risk population.

In this paper a new tool to assess viscoelastic and dielectric properties of human fluids is presented. Shear horizontal polarized surface acoustic waves (SH-SAW) are used to detect the viscoelastic properties of coagulating blood and blood plasma samples. One-port SAW resonators, with fundamental modes of 85, 170 und 340 MHz were developed. Additionally, their electrode structures can be used simultaneously to detect the dielectric behavior of the whole system by impedance spectroscopy while the frequency ranges from kHz to MHz. The combination of both methods offers the detection of clinical relevant blood parameters like the blood coagulation time and the hematocrit value within one measurement.

Background: Bernard-Soulier syndrome (BSS) is a severe congenital bleeding disorder characterized by thrombocytopenia, thrombocytopathy and decreased platelet adhesion. BSS results from genetic alterations of the glycoprotein (GP) Ib/IX/V complex. Methods: We report on a patient demonstrating typical BSS phenotype (thrombocytopenia with giant platelets, bleeding symptoms). However, BSS was not diagnosed until he reached the age of 39 years. Results: Flow cytometry of the patient’s platelets revealed absence of GPIb/IX/V receptor surface expression. In addition, immunofluorescence analysis of patient’s platelets demonstrated very faint staining of GPIX. A novel homozygous deletion comprising 11 nucleotides starting at position 1644 of the GPIX gene was identified using molecular genetic analysis. Conclusions: The novel 11-nucleotide deletion (g.1644_1654del11) was identified as causing the bleeding disorder in the BSS patient. This homozygous deletion includes the last 4 nucleotides of the Kozak sequence as well as the start codon and the following 4 nucleotides of the coding sequence. The Kozak sequence is a region indispensable for the initiation of the protein translation process, thus preventing synthesis of functional GPIX protein in the case of deletion. Hintergrund: Das Bernard-Soulier-Syndrom (BSS) ist eine angeborene Blutungsstörung, die mit Thrombozytopenie, Thrombozytopathie und verminderter Thrombozytenadhäsion assoziiert ist. BSS wird durch genetische Veränderungen des Glykoprotein(GP)-Ib/IX/V-Komplexes verursacht. Methoden: Wir berichten über einen Patienten mit typischem BSS-Phänotyp (Thrombozytopenie mit Riesenthrombozyten, Blutungssymptome). Dennoch wurde die Diagnose BSS erst im Alter von 39 Jahren gestellt. Ergebnisse: Die Durchflusszytometrie der Thrombozyten des Patienten ergab eine fehlende Oberflächenexpression des GPIb/IX/V-Rezeptors. Zusätzlich zeigten Immunfluoreszenz-Analysen der Thrombozyten eine nur sehr schwache Anfärbung von GPIX. In der molekulargenetischen Analyse wurde eine noch nicht bekannte homozygote Deletion von 11 Nukleotiden (beginnend an Position 1644 im GPIX-Gen) identifiziert. Schlussfolgerungen: Diese neue Deletion von 11 Nukleotiden (g.1644_1654del11) wurde als Ursache für die vermehrte Blutungsneigung bei dem BSS-Patienten identifiziert. Von der homozygoten Deletion betroffen sind die letzten 4 Nukleotide der Kozak-Sequenz sowie das Startkodon und weitere 4 Nukleotide des kodierenden Bereichs. Die Kozak-Sequenz ist unerlässlich für die Initiation der Translation in der Proteinbiosynthese, so dass die bei dem Patienten nachgewiesene Deletion die Synthese des funktionellen GPIX-Proteins verhindert. Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

It was with great interest that we read the recent recommendations regarding the treatment of acquired hemophilia A published in Haematologica1 and followed the subsequent discussion.2,3 The described treatment algorithm for this disease, which occurs at an incidence of 1.5/million/year,4 stresses the importance of early eradication of the acquired fVIII-inhibitor. Using prednisolone alone or in combination with cyclophosphamide, this approach will be successful in 30–50% of affected individuals.5 As Huth-Kuhne pointed out, the CD20-antibody rituximab may be an attractive alternative in non-responders or patients with contraindications to steroids.6 However, concerns have been raised because of rituximab side-effects including associations with progressive multifocal leukencephalopathy (PML).7 Furthermore, as Mannucci et al.2 emphasized, the use of this drug is hampered by its substantial costs. Restricting the administered dose to the lowest effective level, seems to be a reasonable strategy to minimize potential risks and costs of rituximab treatment. We report a 66-year old male AHA-patient successfully treated with single low-dose rituximab. The patient presented with multiple hematomas and severe anemia after recovering from a respiratory infection. Prolonged activated partial-thromboplastin-time (aPTT) in combination with undetectable fVIII-activity due to an fVIII-inhibitor was observed. There was no clinical history of any prior hemorrhages nor a family history of bleeding. Clinical and imaging examinations could not reveal any pathological findings suggesting an idiopathic cause of AHA. Prednisolone (1 mg/kg) was started immediately and 2 units of packed red blood cells had to be administered. Afterwards, bleeding ceased, correlating with an increase in fVIII-activity to 28%. Steroids were completely tapered over the next weeks and the patient was lost to follow-up. However, during the following year the patient experienced recurrent episodes of minor bleeding. Seventeen months after the first onset of AHA, he again presented at our institution for follow-up. He reported no spontaneous bleeding episodes; however, fVIII-activity was 15% translating into an fVIII-inhibitor of 133 BU (Bethesda Units). Two months later, severe limb-bleeding requiring surgical treatment occurred after a minor accident. FVIII-inhibitor could still be detected at high levels (79 BU). Because of impaired wound-healing, steroids were deemed contraindicated. Therefore, given our experience with patients with auto-immune diseases other than AHA,8 inhibitor-eradication treatment was started with a single flat dose of 100 mg rituximab. Treatment was well tolerated and three weeks later complete B-cell depletion could be demonstrated (Figure 1). There was no reoccurrence of significant bleeding and even an accident was tolerated without complications. At last presentation, nearly ten months after rituximab treatment, the patient had only a low residual fVIII-inhibitor activity, in spite of a normal B-cell count. FVIII-levels stabilized at a level of 15 to 20% and aPTT was never again found to be prolonged after rituximab treatment. Figure 1. Clinical course overview. Time since diagnosis in months (x-axis). fVIII-inhibitor titer in Bethesda Units (BU) (left y-axis). B-cell proportion (%) (right y-axis). R: time of rituximab application. The clinical course of our patient demonstrates that complete B-cell depletion can be achieved with low-dose rituximab in AHA. We are aware that given the persistently low fVIII-level, our patient does not meet the strict criteria for inhibitor eradication proposed by Huth-Kuhne. On the other hand, we have to acknowledge that he experienced no further bleeding and had inhibitor-titers near the detection limit. We, therefore, believe that future clinical trials should incorporate this low-dose regimen rather than the classical lymphoma-dose (e.g. 375 mg/m2) in the treatment of acquired hemophilia A.

In our previous work we demonstrated the general suitability of surface and bulk acoustic wave (SAW and BAW) structures for recording the continuous change of fluidic behavior in the course of blood coagulation processes [Guhr et al.,2005]. Special advantage can be taken from the direct impact of fluid consistence on SAW and BAW device parameters (e. g. resonator frequency, quality factor, impedance). Beside the ability of continuous detection of coagulation process further tests can be realized with SAW and BAW devices as was shown in an acoustic version of thrombelastography [Guhr et al.,2006]. In this paper, we make the proposal to describe the observed features of blood in the frame of non-Newtonian behavior. Therefore we distinguish between acoustic monitoring of shear viscosity and shear elasticity changes during the whole process of haemostasis. The goal is to get more detailed insight into the ongoing biological process. That approach is suggested from modeling of resonance-antiresonance behavior of thickness shear-mode resonators (TSM), also called quartz crystal microbalance (QCM). The response of resonance and antiresonance parameters can be significantly different from each other, depending on the shear viscosity and elasticity of the loaded fluid. For example, the frequency shifts of both resonances have the same or the opposite sign when changing the viscosity or the elasticity, respectively. The results of modeling the QCM response to viscoelastic loading are used for the interpretation of time dependent measurement signals of the coagulation process.

Background The risk for venous thromboembolism after long-haul flights represents a controversial issue. The aim of our study was to assess the incidence of venous thrombosis associated with long-haul flights in a prospective, controlled cohort study. Methods We included 964 passengers returning from long-haul flights (flight duration, ≥8 hours) and 1213 nontraveling control subjects. We excluded participants who were being treated with anticoagulant drugs or who used compression stockings. Main outcome measures were the incidence of ultrasonographically diagnosed thrombosis in the calf muscle and deep veins, symptomatic pulmonary embolism, and death. Results We diagnosed venous thrombotic events in 27 passengers (2.8%) and 12 controls (1.0%) (risk ratio [RR], 2.83; 95% confidence interval [CI], 1.46-5.49). Of these, 20 passengers (2.1%) and 10 controls (0.8%) presented with isolated calf muscle venous thrombosis (RR, 2.52; 95% CI, 1.20-5.26), whereas 7 passengers (0.7%) and 2 controls (0.2%) presented with deep venous thrombosis (RR, 4.40; 95% CI, 1.04-18.62). Symptomatic pulmonary embolism was diagnosed in 1 passenger with deep venous thrombosis ( P = .44). All of these individuals had normal findings at baseline ultrasonography. Passengers with isolated calf muscle venous thrombosis or deep venous thrombosis had at least 1 risk factor for venous thrombosis (>45 years of age or elevated body mass index in 21 of 27 passengers). The follow-up after 4 weeks revealed no further venous thromboembolic event. Conclusions Long-haul flights of 8 hours and longer double the risk for isolated calf muscle venous thrombosis. This translates into an increased risk for deep venous thrombosis as well. In our study, flight-associated thrombosis occurred exclusively in passengers with well-established risk factors for venous thrombosis.

The risk of venous thromboembolism associated with long-haul flights is the subject of controversy. In a prospective, controlled study, we examined 160 passengers before and after return from a long-haul flight and 160 age-matched and sex-matched, non-travelling volunteers using venous compression ultrasound. Deep vein thrombosis was not observed in either group. Isolated calf muscle vein thrombosis (ICMVT) was present in 4/160 (2.5%) flight passengers and in 1/160 (0.6%) controls. All subjects with ICMVT were clinically asymptomatic, and ICMVT was located in the soleal muscle veins in all four subjects. Three of the four passengers with ICMVT had other risk factors for thrombosis.

Background At the end of VTE treatment, increasing D-Dimer levels after discontinuation of Vitamin-K antagonists (VKA) have been shown to indicate coagulation activation and increased risk of VTE recurrence. However, it is unknown if changes of coagulation activation parameters will be similar after discontinuation of direct oral anticoagulants (DOAC) such as apixaban, rivaroxaban and dabigatran. Furthermore, the clinical impact of these changes is still unclear. Objectives To quantify changes of coagulation activation parameters at the end of VTE treatment with VKA or DOAC and to evaluate their positive predictive value for VTE recurrence at 12 months. Patients and Methods Blood samples for coagulation tests were collected from consenting patients with proximal VTE who discontinued anticoagulation treatment at the end of apixaban, dabigatran or rivaroxaban phase-III VTE treatment trials. Furthermore, similar samples were obtained from VKA patients at the end of treatment. From all patients, samples for D-dimer (DD), prothrombin fragments (F1+2) and thrombin-antithrombin complexes (TAT) measurements were collected at the end of treatment and 4 weeks later. Samples were analysed by blinded lab personnel and statistically evaluated for differences between VKA and DOAC regarding changes between both samples as well as absolute values at 4 weeks. Finally, all patients underwent 12 months follow-up by phone calls to establish rates of recurrent VTE or death from any cause. Results Blood samples were obtained from patients discontinuing apixaban (A; n=37), dabigatran (D; n=17), rivaroxaban (R; n=9) and VKA (n=184), respectively. Absolute values and relative changes of DD, F1+2 and TAT at baseline and 4 weeks were not significantly different between VKA or the DOAC cohorts. Irrespective of the anticoagulant treatment, DD and F1+2 but not TAT demonstrated a significant increase between baseline and week 4 (figure 1). At 12 months, 18 patients (7.3%) had recurrent VTE and 2 patients (0.8%) were dead. Regarding clinical outcomes at 12 months, the negative predictive values (NPV) of DD, F1+2 and TAT were highest for patients after VKA treatment (at least 0.93) and systematically lower for DOAC patients (ranging between 0.86 and 0.91). In contrast, positive predictive values (PPV) of DD, F1+2 and TAT were systematically higher in DOAC patients (0.19 to 0.43) compared to VKA patients (0.03-0.16) with highest values for TAT-complexes > 200% baseline (PPV VKA 0.14; PPV DOAC 0.43), which was also seen in logistic regression analysis with a significant risk increase for VTE/death (Odds ratio for TAT > 200% baseline 5.0; p=0.006). None of the other parameters showed a correlation to the risk of recurrent VTE or death. Conclusion Changes of DD, F1+2 and TAT values post treatment are not different between patients discontinuing VTE treatment with VKA, apixaban, dabigatran or rivaroxaban. NPV of DD, F1+2 and TAT for recurrent VTE/death are higher in VKA than DOAC patients, while PPV are higher in DOAC patients. At 4 weeks, a TAT increase over 200% of baseline value was found to be associated with a 5-fold increase of recurrent VTE or death with a PPV of 0.14 for VKA patients and of 0.43 for DOAC patients. Disclosures: Werth: Bayer Healthcare: Honoraria. Beyer-Westendorf:Bayer Healthcare: Research Funding, Speakers Bureau; Boehringer Ingelheim: Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau.

The accumulation of triglyceride-rich lipoproteins is an independent factor for an increased risk for premature arteriosclerosis. Common mutations in the lipoprotein lipase (LPL) gene are at least in part inherited susceptibility factors involved in the age- and sex-dependent phenotypic expression of hypertriglyceridemia. It can be estimated that about 20% of patients with hypertriglyceridemia are carriers of common LPL gene mutations (Asp9Asn, Asn291Ser, Trp86Arg, Gly188Glu, Pro207Leu, Asp250Asn) associated with the HLP. Genotyping of these LPL gene mutations is recommended especially in patients with high risk for premature arteriosclerosis. A comparably high number of individuals are carriers of common mutations (Ser447X) or silent mutations (Thr361) associated with low favorable lipids.

In this study we investigated a group of patients in whom a resistance to APC (activated protein C) was found but no Leiden mutation existed in the presence of missense mutations in the first 1200 bp of the Exon 13 (B-domain) in the factor V (FV) gene. The determination of the APC response was performed using the Immunochrom(R) APC response Test Kit. The mutations were determined by temperature gradient gel electrophoresis and DNA sequencing. In the APC-resistant patients without the FV Leiden, we found 4 silent mutations (2298CT, 2325TC, 2379AG, 2391AG) and 4 missense mutations (2540AC, 2663AG, 2684AG, 2863AG), which code for the amino acids N789T (GenBank Accession # AF119360), K830R, H837R, and K897E. In all of the patients and controls, the polymorphisms at nucleotide positions 2391, 2663, 2684, and 2863 appeared to be associated. In the major allele all bases are A (A allele) and in the minor allele are G (G allele). A significantly lower G allele frequency was observable in the patient group than in the control group (0.14 vs. 0.31; p0.05). The frequency of the 2540C allele, which is associated with the 2379G and the 4070G allele (non-Leiden!), did not differ significantly between the patient and the control groups. We suggest that the G allele, which is not associated with the FV Leiden mutation, as well as the [2379G; 2540C; 4070G] allele have no influence on the APC cofactor function itself, or only subtly as determined in the test systems used.

The growth factor bound protein GRB2, a 25-kDa cytosolic protein, plays a key role in two separate pathways of the insulin signal transduction system leading from the insulin receptor to the Ras proteins and thus affecting mitogenic signaling. GRB2 regulates Ras activation through association with the guanine nucleotide exchange factor Sos. The GRB2/Sos complex can connect with insulin receptor substrate 1 (IRS-1), which is one of the primary targets of the insulin and insulin-like growth factor receptors. In a second pathway, independent of IRS-1, GRB2 links the insulin receptor to Ras signaling through another adapter protein, called Shc. In protooncogenic and other noninsulin signaling systems, GRB2 appears to link receptor tyrosine kinases to Ras in similar pathways as well. This study presents the exon-intron organization of the human GRB2 gene. After primers were placed across the known mRNA sequence, Long PCR products spanning introns and their adjacent splice sites were amplified and adequately sequenced to establish the splice sites and flanking regions. The gene was found to consist of five exons (ranging from 78 to 186 bp) and of four introns (from approximately 1 to approximately 7 kb). Intron primers for the respective exons were generated using the newly found flanking sequences. All exons were successfully amplified and sequenced, and the data obtained from Long PCR sequencing were confirmed.

LDL apheresis is highly efficient in reducing elevated plasma cholesterol. Due to strict indications only patients with severe, refractory hypercholesterolemia are treated with this method. Mutations in the LDL receptor gene are major genetic causes for severe hypercholesterolemia. Screening the entire gene in LDL apheresis patients from Saxony should determine whether an increased frequency of defined mutations is responsible for the atherogenic hypercholesterolemia in this group. 31 unrelated patients (15 male, 16 female, age 33-71 yrs.) were included in the analysis. The LDL-R gene was screened using SSCP and/or automated sequencing. The familial defective apolipoprotein B-100 (FDB) mutation was genotyped using established PCR techniques. Nineteen of 31 patients were carriers of an LDL-R mutation. Ten missense and two nonsense mutations, three insertions and two deletions were detected. The mutations C74S, C74R, T87M, 660delC, 662insCCCCG, 680insGGACAAATCTGA, 1428insC and 2167delG have not been previously described. One patient was compound heterozygous for two missense mutations. Two further patients were heterozygous for FDB. No mutations were found among controls. A genetic background for hypercholesterolemia in the LDL-R could be established in about 61% of the patients examined. Therefore, methods of DNA analysis allow to recognize and adequately treat a large portion of high-risk individuals at an early stage. © 2000 Wiley-Liss, Inc.