Posts Tagged ‘Dr. Venkat S. Karra’

Author: Dr. Venkat S. Karra, Ph.D.

Platelets are a natural source of growth factors and they circulate in the blood. They are involved in hemostasis, leading to the formation of blood clots. Platelets, otherwise known as thrombocytes, are small, irregularly shaped clear cell fragments derived from fragmentation of precursor megakaryocytes. The average lifespan of a platelet is 5 to 9 days. An abnormality or disease of the platelets leads to a condition called thrombocytopathy.

For example:
1. If the number of platelets is too low (called thrombocytopenia), excessive bleeding can occur.

Disorders leading to a reduced platelet count are:
Idiopathic thrombocytopenic purpura – also known as immune thrombocytopenic purpura (ITP)
Thrombotic thrombocytopenic purpura
Drug-induced thrombocytopenic purpura (for example heparin-induced thrombocytopenia (HIT))
Gaucher’s disease
Aplastic anemia
Alloimmune disorders
Fetomaternal alloimmune thrombocytopenia

2. If the number of platelets is too high (called thrombocytosis), blood clots (thrombosis) can form. Such clots in the blood may obstruct blood vessels and result in events like stroke, myocardial infarction, pulmonary embolism or the blockage of blood vessels to other parts of the body (e.g., arms, legs).

Disorders featuring an elevated count are:
Thrombocytosis, including essential thrombocytosis (elevated counts, either reactive or as an expression of myeloproliferative disease).

3. Thrombasthenia is a condition in which a decrease in function of platelets is observed.

Disorders leading to platelet dysfunction or reduced count are:
HELLP syndrome
Hemolytic-uremic syndrome

Platelets play a significant role in the repair and regeneration of connective tissues. They release a multitude of growth factors, which have been used as an adjunct to wound healing, include:

Platelet-derived growth factor (PDGF), a potent chemotactic agent,
TGF beta, which stimulates the deposition of extracellular matrix.
Fibroblast growth factor,
Insulin-like growth factor 1,
Platelet-derived epidermal growth factor,
Vascular endothelial growth factor.

As said earlier, the function of platelets is the maintenance of hemostasis (the opposite of hemostasis is hemorrhage). This is achieved primarily by the formation of thrombi. When a damage to the endothelium of blood vessels occurs, the endothelial cells stop secretion of coagulation and aggregation inhibitors and instead secrete von Willebrand factor which initiate the maintenance of hemostasis after injury.

Hemostasis has three major steps: 1) vasoconstriction, 2) temporary blockage of a break by a platelet plug, and 3) blood coagulation, or formation of a clot that seals the hole until tissues are repaired.

The platelets get activated when a damage occurs to the blood vessel and the platelets clump at the site of blood vessel injury as a protective mechanism – a process that precedes the formation of a blood clot. This is the case if there is a damage to the endothelium otherwise thrombus formation should be considered seriously and must be inhibited immediately.

Vascular spasm is the first response as the blood vessels constrict to allow less blood to be lost during the injury to the blood vessel. In the second step – platelet plug formation – platelets stick together to form a temporary seal to cover the break in the vessel wall. The third and last step is called coagulation or blood clotting. Coagulation reinforces the platelet plug with fibrin threads that act as a “molecular glue”

Disorders of platelet adhesion or aggregation are:
Bernard-Soulier syndrome
Glanzmann’s thrombasthenia
Scott’s syndrome
von Willebrand disease
Hermansky-Pudlak Syndrome
Gray platelet syndrome

In normal hemostasis a thin layer of endothelial cells, that are lined with the inner surface of blood vessels, act to inhibit platelet activation by producing nitric oxide, endothelial-ADPase (which clears away the platelet activator, ADP – this activator otherwise can be blocked by the famous blockbuster clopidogrel), and PGI2 (also known as prostacyclin or eicosanoids, like PGD2, PGI2 is an inflammatory product that inhibits the aggregation of platelets). Intact blood vessels are central to moderating blood’s tendency to clot because the endothelial cells of intact vessels prevent blood clotting with a heparin-like molecule and thrombomodulin and prevent platelet aggregation with
1. Nitric oxide (NO), and
2. Prostacyclin (PGI2) – a member of eicosanoids family.

In this post, nitric oxide role in inhibiting platelet aggregation will be presented. Similarly Interaction of NO and prostacyclin (PGI2) in vascular endothelium will be presented as a separate post.

Nitric oxide (NO) and its role in inhibiting platelet aggregation:

Nitric oxide (NO) is known as the ‘endothelium-derived relaxing factor’, or ‘EDRF’. The endothelium (inner lining) of blood vessels uses NO to signal the surrounding smooth muscle to relax, thus resulting in vasodilation and increasing blood flow. NO is biosynthesized endogenously from L-arginine, oxygen and NADPH by various nitric oxide synthase (NOS) enzymes. Nitric oxide is highly reactive and yet diffuses freely across membranes that makes it ideal for a transient paracrine (between adjacent cells) and autocrine (within a single cell) signaling molecule.

This is an important cellular signaling molecule involved in many physiological and pathological processes. It is a powerful vasodilator with a short half-life of a few seconds in the blood. Low levels of nitric oxide production are important in protecting organs such as the liver from ischemic damage. Nitric oxide is considered an antianginal drug as it causes vasodilation, which can help with ischemic pain, known as angina, by decreasing the cardiac workload. By dilating the veins, nitric oxide lowers arterial pressure and left ventricular filling pressure. This vasodilation does not decrease the volume of blood the heart pumps, but rather it decreases the force the heart muscle must exert to pump the same volume of blood.

Chronic expression of NO is associated with various carcinomas and inflammatory conditions including Type-1 diabetes, multiple sclerosis, arthritis and ulcerative colitis.

Endothelium-derived relaxing factor (EDRF), the best-characterized is nitric oxide (NO), is produced and released by the endothelium to promote smooth muscle relaxation. EDRF was discovered and characterized by Robert F. Furchgott, a winner of the Nobel Prize in Medicine in 1998 with his co-researchers Louis J. Ignarro and Ferid Murad.

According to Furchgott’s website at SUNY Downstate Medical Center, “…we are investigating whether the endothelium-derived relaxing factor (EDRF) is simply nitric oxide or a mixture of substances”.

Although there is strong evidence that nitric oxide elicits vasodilation, there is some evidence tying this effect to neuronal rather than endothelial reactions.

The article says that “The possibility that neuronal rather than endothelial production of NO might play a significant role in the aetiology of essential hypertension is a promising area for future human research”.

Mechanism of Platelet Aggregation:

Platelets aggregate, or clump together, using fibrinogen and von Willebrand factor (vWF) as a connecting agent. The most abundant platelet aggregation receptor is glycoprotein IIb/IIIa (gpIIb/IIIa) which is a calcium-dependent receptor for fibrinogen, fibronectin, vitronectin, thrombospondin, and vWF. Other receptors include GPIb-V-IX complex (vWF) and GPVI (collagen).

Activated platelets will adhere, via glycoprotein (GP) Ia, to the collagen that is exposed by endothelial damage. Aggregation and adhesion act together to form the platelet plug. Myosin and actin filaments in platelets are stimulated to contract during aggregation, further reinforcing the plug. Platelet aggregation is stimulated by ADP, thromboxane, and α2 receptor-activation, and further enhanced by exogenous administration of anabolic steroids.

In an injury to the blood vessel, once the blood clot takes control of the bleeding, the aggregated platelets help the healing process by secreting chemicals that promote the invasion of fibroblasts from surrounding connective tissue into the wounded area to completely heal the wound or form a scar. The obstructing clot is slowly dissolved by the fibrinolytic enzyme, plasmin, and the platelets are cleared by phagocytosis.

Possible usefulness of measuring GP IIb-IIIa content as a marker of increased platelet reactivity is discussed in the following very recent (2011) reveiw article: “Glycoprotein IIb-IIIa content and platelet aggregation in healthy volunteers and patients with acute coronary syndrome”.

Further readings:

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Curated by: Dr. Venkat S. Karra, Ph.D.

A human brain showing frontotemporal lobar deg...

The number of patients with dementia have been increasing exponentially with the aging of society.  The development of AD research has clarified that the pathogenesis of AD is initiated by amyloidosis with secondary tauopathy and provided a strategy for investigating drugs that may improve or cure AD.

Mild cognitive impairment (MCI) as a prodromal stage of AD and the pathogenesis of Dementia with Lewy bodies (DLB) and Frontotemporal lobar degeneration (FTLD) as a non-AD type dementia have also been elucidated. Currently, a consortium study by the Alzheimer Disease Neuroimaging initiative (ADNI) is being performed to establish global clinical evidence regarding a neuropsychiatric test battery, CSF biomarkers, neuroimaging including MRI, FDG-PET, and amyloid PET to predict progression from MCI to AD and to promote studies of basic therapy for AD [1].

Several new biomarkers such as Aβ oligomer, α-synuclein, and TDP-43 are now under investigation for further determination of their usefulness to detect AD and other non-AD type dementia.

Cerebrospinal Fluid Aβ40, Aβ42, Tau, and Phosphorylated Tau biomarkers have been used for a clinical diagnosis of AD, discrimination from the Vascular dementia (VaD) and non-AD type dementia, exclusion of treatable dementia and MCI, prediction of AD onset and evaluation of the clinical trials of an anti-Aβ antibody, Aβ vaccine therapy, and secretase inhibitors [2–4].

In the current study Schoonenboom et al., [10] conducted a large cohort of patients with different types of dementia to determine how amyloid β 42 (Aβ42), total tau (t-tau), and phosphorylated tau (p-tau) levels behave in CSF.

Aβ is produced mainly in the nerve cells of the brain, and it is secreted about 12 hours later into the CSF, then excreted through the blood-brain barrier 24 hours later into blood (Aβ clearance), and finally degraded in the reticuloendothelial system. Aβ levels are regulated in strict equilibrium among the brain, CSF, and blood [6, 7]. Aβ levels are high while awake and low while a sleep suggesting the presence of a daily change in the CSF Aβ amounts and it is because Aβ amounts in CSF are controlled by orexin and thus collection of CSF by lumbar puncture early in morning in a fasting state is recommended [5].

In AD brains, Aβ42 forms insoluble amyloids and accumulates as insoluble amyloid fibrils in the brain. The reason Aβ42 levels are decreased in the CSF of AD patients is considered to be caused by deterioration of physiologic Aβ clearance into the CSF in AD brains [2, 3]. CSF total tau levels increase slightly with aging. However, CSF tau levels show a 3-fold greater increase in AD patients than in normal controls [8].

It is thought that the rise in CSF total tau is related to degeneration of axons and neurons and to severe destructive disease of the nervous system. Several diseases show slightly increased tau levels such as VaD, multiple sclerosis, AIDS dementia, head injury, and tauopathy. However, CSF tau levels show significant increases in Creutzfeldt-Jakob disease (CJD) and meningoencephalitis [8].

These biomarkers can be measured with an Amyloid ELISA Kit (Wako), which is commercially available and used worldwide. The ELISA kit was developed in Japan by Suzuki et al. and shows extremely high sensitivity and reproducibility [9]. INNOTEST β-AMYLOID1-42 (Innogenetics), for Aβ42 is used widely in Europe and America.

Several assay kits for total tau and phosphorylated tau are also used for the measurement of CSF tau. Currently, total tau is measured using INNOTEST hTau Ag (Innogenetics). There are 3 ELISA systems for measurement of phosphorylated tau that recognize the special phosphorylation sites at Ser199 (Mitsubishi Chemical Corp.), Thr181 (Innogenetics) and Thr231 (Applied NeuroSolutions Inc.), and phosphorylated tau levels are increased in CSF of AD on assays using these kits. Of these 3 kits, INNOTEST PHOSPHO-TAU (181) (Innogenetics) is commercially available and used widely. Recently, INNO-BIA AlzBio3 by Innogenetics has been able to measure Aβ1-42, total tau, and P-tau181P simultaneously in 75 μL of CSF, which is a very small amount of CSF.

In the current study researchers used the following strategy to collect Baseline CSF and Aβ42, t-tau, and p-tau (at amino acid 181) were measured in CSF by ELISA:

Types of patients with Alzheimer disease (AD) = 512 patients
Types of patients with other types of dementia (OD) = 272 patients
Types of patients with a psychiatric disorder (PSY) = 135 patients
Types of patients with subjective memory complaints (SMC) = 275 patients
Autopsy was obtained in a subgroup of about 17 patients.

The study suggested that CSF Aβ42, t-tau, and p-tau are useful in differential dementia diagnosis, whereas in DLB, FTLD, VaD, and CBD, a substantial group exhibited a CSF AD biomarker profile, which requires more autopsy confirmation in the future.

The study found a correct classification of patients with AD (92%) and patients with OD (66%)  when CSF Aβ42 and p-tau were combined.
Patients with progressive supranuclear palsy had normal CSF biomarker values in 90%.

Patients with Creutzfeldt-Jakob disease demonstrated an extremely high CSF t-tau at a relatively normal CSF p-tau.

CSF AD biomarker profile was seen in

47% of patients with dementia with Lewy bodies (DLB),

38% in corticobasal degeneration (CBD), and

30% in frontotemporal lobar degeneration (FTLD) and vascular dementia (VaD).

PSY and SMC patients had normal CSF biomarkers in 91% and 88%.

Older patients are more likely to have a CSF AD profile.

Concordance between clinical and neuropathologic diagnosis was 85%.

CSF markers reflected neuropathology in 94%.

The study concluded that CSF Aβ42, t-tau, and p-tau are useful in differential dementia diagnosis. However, in DLB, FTLD, VaD, and CBD, a substantial group exhibit a CSF AD biomarker profile, which requires more autopsy confirmation in the future.


1. R. C. Petersen, P. S. Aisen, L. A. Beckett et al., “Alzheimer’s Disease Neuroimaging Initiative (ADNI): clinical characterization,” Neurology, vol. 74, no. 3, pp. 201–209, 2010.

2. M. Shoji and M. Kanai, “Cerebrospinal fluid Aβ40 and Aβ42: natural course and clinical usefulness,” Journal of Alzheimer’s Disease, vol. 3, no. 3, pp. 313–321, 2001.

3. M. Shoji, M. Kanai, E. Matsubara et al., “The levels of cerebrospinal fluid Aβ40 and Aβ42(43) are regulated age-dependently,” Neurobiology of Aging, vol. 22, no. 2, pp. 209–215, 2001.

4. M. Kanai, E. Matsubara, K. Isoe et al., “Longitudinal study of cerebrospinal fluid levels of tau, Aβ1-40, and Aβ1-42(43) in Alzheimer’s disease: a study in Japan,” Annals of Neurology, vol. 44, no. 1, pp. 17–26, 1998.

5. J. E. Kang, M. M. Lim, R. J. Bateman et al., “Amyloid-β dynamics are regulated by orexin and the sleep-wake cycle,” Science, vol. 326, no. 5955, pp. 1005–1007, 2009.

6. M. Shoji, T. E. Golde, J. Ghiso et al., “Production of the Alzheimer amyloid β protein by normal proteolytic processing,” Science, vol. 258, no. 5079, pp. 126–129, 1992.

7. R. J. Bateman, E. R. Siemers, K. G. Mawuenyega et al., “A γ-secretase inhibitor decreases amyloid-β production in the central nervous system,” Annals of Neurology, vol. 66, no. 1, pp. 48–54, 2009.

8. M. Shoji, E. Matsubara, T. Murakami et al., “Cerebrospinal fluid tau in dementia disorders: a large scale multicenter study by a Japanese study group,” Neurobiology of Aging, vol. 23, no. 3, pp. 363–370, 2002.

9. N. Suzuki, T. T. Cheung, X. D. Cai et al., “An increased percentage of long amyloid β protein secreted by familial amyloid β protein precursor (βAPP) mutants,” Science, vol. 264, no. 5163, pp. 1336–1340, 1994.


10. N.S.M. Schoonenboom et al., Cerebrospinal fluid markers for differential dementia diagnosis in a large memory clinic cohort

For further insight read the following excellent review article by M. Shoji

Biomarkers of Dementia

Special thanks to Wikipedia for excellent relevant pictures and keyword links.

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