Design,development and characterization of ACT017, a humanized Fab that blocks platelet's glycoprotein VI function without causing bleeding risks

Glycoprotein VI is a platelet-specific collagen receptor critical for in vivo formation of arterial thrombosis. It is also considered as an attractive target for the development of anti-thrombotic drugs because blocking glycoprotein (GP)VI inhibits platelet aggregation without inducing detrimental effects on physiologic hemostasis.

Here, we present data on the identification, in vitro and ex vivo pharmacology of a humanized Fab fragment designated as ACT017. ACT017 was selected out of 15 humanized variants based upon structural and functional properties. It was produced under GMP-like conditions followed by detailed physico-chemical analysis and functional characterization indicating high antigen-binding specificity and affinity. In addition, we demonstrate, in a dose-escalation study, that ACT017 has a high capacity to specifically inhibit collagen-induced platelet aggregation ex vivo after injection to the macaque without inducing thrombocytopenia, GPVI depletion or bleeding side effects as is the case for conventional anti-platelets. Therefore, ACT017 is a promising therapeutic candidate for the development of a new generation of safe and efficient anti-thrombotic drugs.     

血浆D- 二聚体检测在下肢深静脉血栓诊断中的价值

目的:探讨血浆D-二聚体在下肢深静脉血栓形成(DVT)中的诊疗价值。方法:用SYS MEX CA2 1500全自动血凝分析仪检测185例可疑为DVT患者及60例健康体检者血浆中纤维蛋白降解产物D-二聚体水平,比较其D-二聚体水平与健康体检组的差别。结果:60例健康体检者及185例疑似DVT患者中18例患者血浆D-二聚体含量<临界值,经下肢彩色多普勒超声检查证实无一例患有DVT;167例患者血浆D-二聚体含量≥临界值,经下肢彩色多普勒超声检查证实DVT患者150例,D-二聚体检测DVT的敏感性、特异性、阳性预期值、阴性预期值分别为100%、51.43%、89.82%、100%。结论:血浆D-二聚体检测具有快速、经济、无创、可动态监测等优点,可以作为DVT诊断的排除试验,值得在临床检验诊断中推广应用。     

Proteomic analysis of venous thromboembolism

Venous thrombosis has a multicausal basis, and is characterized by a multifaceted combination of inherent phenotypic complexity and genetic predisposition, with acquired and triggering factors further and acutely influencing the likelihood of an individual experiencing a clinically significant thrombotic event. Traditional coagulation tests, especially clot-based assays, are useful for describing major abnormalities in the hemostatic response, but fail in their application to assessing thrombotic risk in the healthy population. Recent evidence also attests that the analysis of a vast array of genes can only explain part of the individual thrombotic risk. Thus, proteomic analysis may hold promise for characterizing or understanding biological pathways and pathophysiological interactions, for improving the diagnosis and identifying novel therapeutic approaches to this prevalent and life-threatening disorder. Herein, we present and discuss available data on proteomic analysis of venous thromboembolism, concluding that further studies supported by high-throughput techniques should be undertaken to elucidate or understand biological pathways and pathophysiological interactions.     

Biomechanics of haemostasis and thrombosis in health and disease: from the macro‐ to molecular scale

Although the processes of haemostasis and thrombosis have been studied extensively in the past several decades, much of the effort has been spent characterizing the biological and biochemical aspects of clotting. More recently, researchers have discovered that the function and physiology of blood cells and plasma proteins relevant in haematologic processes are mechanically, as well as biologically, regulated. This is not entirely surprising considering the extremely dynamic fluidic environment that these blood components exist in. Other cells in the body such as fibroblasts and endothelial cells have been found to biologically respond to their physical and mechanical environments, affecting aspects of cellular physiology as diverse as cytoskeletal architecture to gene expression to alterations of vital signalling pathways. In the circulation, blood cells and plasma proteins are constantly exposed to forces while they, in turn, also exert forces to regulate clot formation. These mechanical factors lead to biochemical and biomechanical changes on the macro‐ to molecular scale. Likewise, biochemical and biomechanical alterations in the microenvironment can ultimately impact the mechanical regulation of clot formation. The ways in which these factors all balance each other can be the difference between haemostasis and thrombosis. Here, we review how the biomechanics of blood cells intimately interact with the cellular and molecular biology to regulate haemostasis and thrombosis in the context of health and disease from the macro‐ to molecular scale. We will also show how these biomechanical forces in the context of haemostasis and thrombosis have been replicated or measured in vitro.     

Multispectral optoacoustic tomography of muscle perfusion and oxygenation under arterial and venous occlusion: A human pilot study

Perfusion and oxygenation are critical parameters of muscle metabolism in health and disease. They have been both the target of many studies, in particular using near‐infrared spectroscopy (NIRS). However, difficulties with quantifying NIRS signals have limited a wide dissemination of the method to the clinics. Our aim was to investigate whether clinical multispectral optoacoustic tomography (MSOT) could enable the label‐free imaging of muscle perfusion and oxygenation under clinically relevant challenges: the arterial and venous occlusion. We employed a hybrid clinical MSOT/ultrasound system equipped with a hand‐held scanning probe to visualize hemodynamic and oxygenation changes in skeletal muscle under arterial and venous occlusions. Four (N = 4) healthy volunteers were scanned over the forearm for both 3‐minute occlusion challenges. MSOT‐recorded pathophysiologically expected results during tests of disturbed blood flow with high resolution and without the need for contrast agents. During arterial occlusion, MSOT‐extracted Hb‐values showed an increase, while HbO₂‐ and total blood volume (TBV)‐values remained roughly steady, followed by a discrete increase during the hyperemic period after cuff deflation. During venous occlusion, results showed a clear increase in intramuscular HbO₂, Hb and TBV within the segmented muscle area. MSOT was found to be capable of label‐free non‐invasive imaging of muscle hemodynamics and oxygenation under arterial and venous occlusion. We introduce herein MSOT as a novel modality for the assessment of vascular disorders characterized by disturbed blood flow, such as acute limb ischemia and venous thrombosis.     

Label-free high frame rate imaging of circulating blood clots using a dual modal ultrasound and photoacoustic system

Deep vein thrombosis (DVT) is a disorder when a blood clot (thrombus) is formed in one of the deep veins. These clots detach from the original sites and circulate in the blood stream at high velocities. Diagnosing these blood clots at an early stage is necessary to decide the treatment strategy. For label-free, in vivo, and real-time detection, high framerate photoacoustic imaging can be used. In this work, a dual modal clinical ultrasound and photoacoustic (PA) system is used for the high framerate PA imaging of circulating blood clots in blood at linear velocities up to 107 cm/sec. Blood clot had 1.4 times higher signal-to-noise ratio (SNR) in the static mode and 1.3 times higher SNR compared to blood PA signal in the flow experiments. This work demonstrates that fast-moving circulating blood clots are easy to recognize against the background PA signal and may aid in early diagnosis.     

Features and incidence of thromboembolic disease: A comparative study between high and low altitude dwellers in Saudi Arabia

To estimate and compare the incidence of thromboembolic disease among patients who are clinically suspected for VTE among high and low altitude dwellers in Saudi Arabia. A prospective study conducted over two years (2011–2013) conducted in two different geographical areas in Saudi Arabia; Abha City and Riyadh City. Patients clinically suspected with deep vein thrombosis and pulmonary embolism was recruited to the study. A detailed social, medical and laboratory investigations were taken from all patients including lifestyle, occupation and smoking. A total of 234 patients participated in the study. There were 146 (62.4%) females and 88 (37.6%) males. Mean age was 51.7 years. A 56.8% incidence of DVT was seen among high altitude dwellers compared to 13.0% among low altitude dwellers. Also, a 12.6% incidence of PE was documented among high altitude dwellers, compared to 4.1% of the low altitude dwellers. VTE was significantly more among high altitude dwellers (81.9%) compared to low altitude dwellers (21.9%). Mean WBC count was significantly higher among the high altitude dwellers (10.8 ± 9.7 vs. 8.2 ± 3.4, p = 0.043). Mean platelet count was significantly higher among the high altitude dwellers compared to the low altitude dwellers (327.4 ± 162.4 vs. 212.0 ± 158.9, p = 0.005). The likelihood of developing VTE is greater among people who resided at moderate to high altitude for prolonged periods of time. The changes in the factors for coagulation including platelet counts may not reflect the true status of hypercoagulability especially if patients have stayed longer in high altitudes because of physiological adaptation to the environment.