Effect of Mouse KOs on Platelet Production and/or Platelet Clearance.
Platelet counts in blood are controlled by the rates of their production by megakaryocytes and the kinetics of their removal. Alterations in either process can lead to thrombocytopenia (TCP) or thrombocytosis. Under conditions of TCP, the spleen and liver are the sites for accelerated platelet destruction, and in thrombocytosis, the spleen can become a supplemental breeding ground for megakaryocytes, in addition to the bone marrow space. Humans produce and remove 1011 platelets per day. Senile or damaged platelets are detected as such and are removed from blood. Platelets must also be removed locally at diverse sites where they have discovered vascular damage, attached and become activated to prevent blood leakage. The mechanism of this local removal in the blood vascular system is not well described or understood. The removal of platelets from flowing blood mandates a system that detects changes that accumulate in the platelet surface with age and that responds by binding and removing the platelets when changes reach a critical level. Surface changes must either increase the availability of clearance ligands or remove anti-clearance signals such as CD47 . Changes in the platelet surface that signal for removal include phosphatidylserine upregulation, deglycosylation of membrane glycoproteins, in particular Gp1bα of the vWf receptor, and Ig binding. Diseases that accelerate removal arbitrate their impact at the platelet surface.
This section highlights recent advancements in understanding how platelet lifetimes in blood are regulated and the discovery of a surprising feedback pathway that links platelet removal by hepatocytes in liver to platelet production in the bone marrow.
2. Sites and cells that remove senile platelets
Platelet senescence is driven by both an internal proteolytic clock and through external changes that occur on the cell surface as they circulate in blood. Internally, platelets have an apoptotic mechanism that sets limits on the overall platelet lifetime at ~10 days . At the time of birth, each mature platelet has a defined quota of the pro-survival protein Bcl-x that with time degrades, releasing its brake on the activities of Bak and Bax that subsequently induce mitochondrial lysis, cytochrome C release, and the activation of cytoplasmic capases. Capases disassemble the cytoskeleton and lead to the upregulation of phosphatidylserine to the platelet surface. Cells that have phosphatidylserine on their surfaces are avidly recognized and removed by the professional phagocytes, macrophages and immature dendritic cells. Multiple receptors have been identified for phosphatidylserine on phagocytes including CLM-1 or CD300f , Tim-4 , BaI1 , or Stabilin-1 receptor .
Platelets express CD47 on their surface. CD47, also called integrin associated protein , is a member of the immunoglobulin superfamily that associates with the integrins, αIIbβ3 and αvβ3, on platelets. A role for CD47 in maintaining platelets in circulation was first recognized in knockout mice, which have platelets counts reduced by ~20% compared to normal. This highly glycosylated surface protein is recognized by the SIRPα transmembrane protein on phagocytes that contains two immunoreceptor tyrosine-based inhibitory domains in its cytoplasmic domain. Hence, ligation by CD47 transmits anti-phagocytic signals to macrophages, independent of the phagocytic receptor engaged, helping to prevent macrophages for engaging phagocytic pathways involving Fc receptors.
One example where phosphatidylserine exposure can induce macrophages to remove platelets occurs with FlnA null platelets. Mice having megakaryocytes and platelets that lack FlnA are macrothrombocytopenic, with platelet counts reduced by 80-90%, compared to WT mice. FlnA null platelets are unstable because they lack FlnA’s actin filament crosslinking activity and its membrane glycoprotein attachment sites, which link the actin cytoskeleton to the plasma membrane. FlnA constitutively binds to the GP1bα chains of the vWF complex, as well as certain β-integrin chains. The high density of the vWfR-FlnA interaction, ~12,000 per platelet, stabilizes the membrane by attaching it to the underlying actin cytoskeleton. Because the vWfR is linked to the sides of actin filaments it also regulates the topology of the vWf receptor, aligning the receptor in linear arrays on the platelet surface. When studied
3. Sialic acid and platelet circulatory lifetime
Sialic acid terminates the N- and O-linked glycans of all cell surface glycoproteins. Desialylation of platelet glycans was the first recognized mechanism that can target platelets for clearance. In 1975, Greenberg and colleagues  showed that desialylation of platelets (and erythrocytes)
The Asgr was one of the first receptors to be identified and characterized  over 40 years ago. Each surface Asgr is a heterotrimer composed of 2 HL-1 chains and 1 HL-2 chain. Knock out animals lacking in the expression of either chain, therefore, do not have functional Asgr receptors on their hepatocytes. The Asgr is a C-type lectin that recognizes exposed β-galactose, the underlying moiety to which sialic acid is linked in carbohydrate chains. Most glycoproteins have their glycans capped by α2,3-linked sialic acid. Galactose exposure is mediated by sialidases present in blood or released into blood by infectious bacteria. Interestingly and of high clinical impact, the Asgr can also bind to α2,6 linked sialic acid residues on glycoproteins.
Desialylated platelets are recognized and removed by the liver Asgr [13-15]. The specific function of the Asgr in platelet removal has been demonstrated by the following evidences: (1) HepG2 cells bind and ingest desialylated platelets
4. Feedback between liver and bone marrow
The importance of hepatocyte-platelet interaction extends beyond simple removal, as the recognition and ingestion of platelets by the Asgr generates cytoplasmic signals in hepatocytes that induce the formation and secretion of cytokines to promote marrow and megakaryocyte growth and maturation. In this case, the key cytokine produced in response to platelet ingestion is thrombopoietin . Thus, the hepatocyte-platelet interaction directly feeds back to megakaryocytes in marrow, helping to stimulate platelet production.
5. Platelet reactive receptors on macrophages
In addition to its receptors that recognize phosphatidylserine, macrophage surfaces are festooned with receptors that can ingest damaged and/or diseased platelets. One group includes the lectin receptors that recognize carbohydrate alterations in platelet glycoproteins. The phagocytic integrin, αMβ2, recognizes and removes chilled and rewarmed platelets that release glycosylases causing β-GlcNAc moieties to expose on N-linked GP1bα glycans. In addition, a second domain on αMβ2 recognizes a different portion of GP1bα . Mannose receptors are a second example of a receptor that detects glycan alterations, recognizing underlying mannose moieties exposed by glycosylases . Fcγ receptors remove Ig-coated platelets from blood and when anti-platelet Igs are present their effectiveness leads to thrombocytopenia.
6. Diseases causing accelerated platelet clearance by macrophages
Accelerated clearance requires either the accumulation of opsonins on the platelet surface such as Igs and complement or the presence of agents in blood that remove protective molecules. Both types of mechanisms occur.
There are many examples of acquired or induced immune thrombocytopenia that cause platelet removal when anti-platelet Igs are generated, deposited on platelets, and are detected by Fcγ receptors on macrophages. These include congenital and drug or pathogen induced thrombocytopenia. In general, platelet clearance is primarily driven by splenic macrophages, a process that can result in splenomegaly. In many cases, patients having ITP, respond well to anti-Fc antibody treatment.
7. Platelet clearance mediated by hepatocytes
Bacterial-derived sialidases, released into blood during sepsis, cause platelet counts to drop precipitously. The target of the blood born bacterial sialidases is sialic acid that caps N-linked glycans on GP1bα, as demonstrated using mice lacking GP1bα , which are resistant to clearance induced by pneumococcal sepsis in WT mice. Cleavage of sialic acid on GP1bα exposes underlying galactose moieties that are recognized by the Asgr . Animals lacking a functional Asgr do not accelerate their platelet clearance in response to sepsis.
A related process accounts for the circulation failure of platelets transfused after rewarming from refrigerated storage. Resting platelets contain sialidases that are stored in an internal compartment that can be released by activation [15, 20]. Rewarming from the cold releases a portion of the sialidase activity to the platelet surface and into the storage media, a process that mediates desialylation of the platelet surface glycoproteins. Since the accelerated clearance of chilled and rewarmed platelets is to a large extend ablated in mice lacking the Asgr (HL-2-/- mice), it is the main receptor that recognizes and removes cold damaged platelets. Ablation of macrophage function in HL-2-/- mice further restores platelet circulation by 15-20%. Hence, macrophages also participate in clearance.
8. Accelerated platelet clearance in Wiskott-Aldrich syndrome
Mutation, truncation and/or deletion of WASp, a protein encoded on the X-chromosome and expressed by blood cells, results in a profound lymphocyte dysfunction that severely compromises the immune system. Severe thrombocytopenia (TCP) is also a signature component of the Wiskott-Aldrich syndrome; WAS platelets are small and have shortened circulatory lifetimes. WAS patients produce diverse autoantibodies and WAS platelets collect higher amounts of surface-associated immunoglobulins (Igs) than do normal platelets . Many human WAS patients respond to splenectomy with increased platelet counts, despite the finding that all patients have been found to be refractory to anti-Fcγ antibodies. Unlike ITP, homologous platelets circulate normally in WAS patients strongly suggesting a more complex mechanism for removal that involves receptors other than Fc.
WASp KO mice have been shown to retain the key features of WAS disease, having T and B lymphocyte dysfunction, enlarged spleens, low platelet counts (70% of normal) and shortened platelet survival times in blood. It has been widely believed that platelet clearance is accelerated in these animals because the autoimmune aspect of the disease results in increased Igs bound to the platelets surface that led to recognition by splenic macrophages. However, as in the human conditions, normal platelets, when transfused into WASp KO mice, circulate normally indicating that a simple anti-platelet antibody mediated clearance is not the mechanism. In mice, splenectomy has been shown to be without effect on the clearance rate.
In efforts to identify the mechanism of removal, WASp Null platelets were transfused into mice lacking specific phagocytic receptors. A survey on macrophage receptors failed to reveal any in which the WASp null platelets had enhanced circulatory lifetimes. However, WASp KO platelets were found to circulate normally in Asgr null mice, a finding once again posits the Asgr as a central molecule involved in the recognition and removal of damaged platelets. The surface of WASp KO platelets is, however, not desialylated and lectin binding studies have instead revealed hypersialylation. This sialylation occurs specifically in the 2,6 linkage, not the normal 2,3 linkage. Critically, the Asgr also receptor recognizes this unique sialic linkage, leading to binding and platelet removal. The carrier of this sialic acid turns out to be surface bound Ig and sialylation of its Fc N-linked glycan shifts recognition of the Fc domain from macrophages to the hepatocytes.
Interestingly, the source of the 2,6 sialyltranferase (ST6Gal1) is liver hepatocytes, which make and secrete this enzyme into blood. This blood enzyme is an acute phase reactant protein, upregulated in liver in response to bacterial sepsis, cancer, or inflammation. In this case, platelet ingestion itself, feedbacks to upregulate ST6Gal1 mRNA transcription and translation and this increases by 3-4 fold the blood levels of this enzyme.
9. Cause of surface alterations that lead to the deposition of Igs on the WASp KO platelet
The modification(s) in WASp KO platelet surface that leads to Ig production by WASp KO B-lymphocytes and Ig-binding are not known. Since WT platelets do not collect Igs in WASp KO plasma or have accelerated clearance in WASp KO animals, surface alterations are specific for the WASp KO platelets. Because WASp is a protein that interacts with the actin cytoskeleton, it is likely that internal cytoskeletal changes in its absence result in an altered topology of platelet receptors or the expression of the neo-epitope. In general, platelet function in the absence of WASp is near normal although as the precision of assays increase, some differences have now been recognized. Active platelets lack small focal actin assembly sites in the absence of WASp, although spreading and filopodial formation are normal. In resting platelets, failure to express WASp alters the stability of microtubules, increasing their acetylation and slowing their turnover. How these internal changes alter the surface remains for future studies.
10. Platelet production
The basic processes involved in megakaryocyte commitment, maturation and platelet formation are well described although many precise details remain to be clarified. Megakaryocytes derive from a renewable population of hematopoietic stem cells that continuously enter the MK/platelet lineage and once committed to produce platelets, live for only a few more weeks before they are converted into hundreds of platelets. Proplatelet and platelet production requires a massive enlargement in MK size that is driven by high levels of mRNA transcription from their amplified polyploid nuclei followed by mRNA translation into platelet essential components. This includes the production of an abundant internal network of membranes called the demarcation membrane system (DMS) that dramatically increases the apparent membrane to surface ratio during proplatelet formation, platelet specific granules, and the synthesis of large amounts of the cytoskeletal machinery that is used to form and fill assembling platelets.
As MKs mature, they develop an extensive network of internal membranes called the DMS that are enriched phosphatidylinositol 4,5 bisphosphate and the vWf receptor  and are used as the primary membrane source for proplatelet elongation. Recent studies by Eckly et al  have begun to shed some light on DMS formation, describing how the DMS forms and matures. To form the DMS, the plasma membrane of megakaryocytes enfolds at specific sites and a perinuclear pre-DMS is generated. Next, the pre-DMS is expanded into its mature form by material added from golgi-derived vesicles and endoplasmic reticulum-mediated lipid transfer. This structural description is in accordance with the studies on platelet glycosyltransferases, which arrive early in the forming DMS and eventually make their way to the megakaryocyte and platelet surfaces . Only a small number of proteins have been identified thus far to participate in the DMS formation process based on alterations in its structure in certain knockout animals. Gross disruptions of this network are found in megakaryocytes isolated from either filamin A knockout or pacsin2 knockout mice, the latter of which connects GP1bα to the actin cytoskeleton and binds pacsin2, a molecule that deforms membrane. The relationship between the DMS and the platelet open canalicular system (OCS) is not clear. The OCS, like the DMS, is a unique anastomosing network of internal membrane tubes that is connected to the plasma membrane at multiple points. The internal canals of the OCS can be identified because they contain vWfR, and hence can be labeled with anti-GP1bα antibodies (Kahr et al).
To release platelets, megakaryocytes in the marrow space move to and nestle the marrow sinusoids where they project their proplatelet protrusions into the blood flow [25, 26]. Whether all proplatelets are directed to grow specifically into the sinusoids or if only some of the proplatelets elaborated by a MK find their way into the sinusoids is unknown, although living MKs in marrow have been observed to have many proplatelet projections, some of which project into the marrow space while others project into the sinusoids . Studies have demonstrated that proplatelet fragments considerably larger than platelets are released by MKs into blood [26, 27] and that proplatelets can be both found, and can mature into platelets, in blood .
The state of our current knowledge of the mechanics of proplatelet production has come primarily from studies on MKs in culture. This work has clarified the essential role of microtubules, which were recognized early as the most prominent structure found within the MK projections  and that proplatelet and platelet production were adversely affected by MT poisons . Subsequent gene expression studies established the importance of β1-tubulin, a tubulin isoform specific for MKs, to the maturation of MKs into proplatelet producing machines . More recent studies using gene deleted MKs have begun to reveal the precise roles of specific proteins in proplatelet and platelet production and these are highlighted below.
Signals that initiate proplatelet formation, if present, remain undefined and it remains possible that the program to make platelets starts when the synthesis of cytoskeletal proteins for this process reaches a critical mass. From a mechanical view, centrosome dissolution precedes proplatelet extension, and the release of MTs from these multiple nucleating sites correlates best with the start of proplatelet elaboration. Released MTs first collect as bundles in the MK cortex where they are driven apart by their associated motor protein, dynein. These MT-dynein reactions deform the membrane outward and generate the structural motor of the proplatelet, which is a MT bundle that folds over in the proplatelet tip and runs back into the shafts. Each bundle is composed of many MTs that are continuously growing and shrinking from their ends. Six types of behaviors characterize the elaboration of proplatelets: elongation, branching, pausing, fusions, fragmentations, and retractions. While the average elongation rate for proplatelets over time is ~1 µm/min, extension normally occurs in bursts and pauses. Burst rates greatly exceed the average rates and under flow, and rates of >30 µm/min have been observed. These rates correlate well with the sliding rates of MTs within the bundles. Pauses in proplatelet extension are not caused by a stoppage in MT sliding, which continues at a 1-6 µm/min rate. Sliding reactions in paused proplatelets appear to build tension into the bundle which when released leads to branching and/or fragmentation. This implies that there are regions within the bundles where MTs are crosslinked to increase resistance or they are pushing against resistive structures. Branching is a modified form of extension derived from tension asymmetry where a portion of the MT bundle detaches from the mother bundle and elongates rapidly forming a new tear shaped structure and proplatelet shaft. Retraction, where the sliding could either reverse or all crosslinking derived tension releases, could serve to subfragment the proplatelets.
In addition to playing a crucial role in proplatelet elongation, the microtubules lining the shafts of proplatelets serve a secondary function — tracks for the transport of membrane, organelles, and granules into proplatelets and assembling platelets at proplatelet ends . Individual organelles are sent from the cell body into the proplatelets, where they move bidirectionally until they are captured at proplatelet ends. Immunofluorescence and electron microscopy studies indicate that organelles are in direct contact with microtubules, and actin poisons do not diminish organelle motion. Therefore, movement appears to involve microtubule-based forces. Bidirectional organelle movement is conveyed in part by the bipolar organization of microtubules within the proplatelet, as kinesin-coated beads move bidirectionally over the microtubule arrays of permeabilized proplatelets. Of the two major microtubule motors — kinesin and dynein — only the plus-end-directed kinesin is situated in a pattern similar to organelles and granules, and is likely responsible for transporting these elements along microtubules. It appears that a twofold mechanism of organelle and granule movement occurs in platelet assembly. First, organelles and granules travel along microtubules and, second, the microtubules themselves can slide bidirectionally in relation to other motile filaments to indirectly move organelles along proplatelets in a “piggyback” fashion
Although microtubules and associated motor and regulatory proteins power proplatelet motility, elimination of certain actin cytoskeletal-associated proteins have now been demonstrated to modulate this process. These include a number of actin-associated proteins, membrane contouring proteins, and signaling proteins.
11. Influence of actin-associated proteins on thrombopoiesis
Since proplatelets elongate, but do not branch, in the presence of the actin disrupting drug cytochalasin B, it is surprising that the deletion of certain actin associated proteins from the megakaryocyte lineage leads to macrothrombocytopenia. It seems likely that the removal of actin modulating proteins, alters and/or increases filamentous actin (F-actin) and cytoskeletal structure to have a dominant inhibitory effect proplatelet maturation and/or platelet release from proplatelets.
12. Actin crosslinking proteins
Three proteins, filamin A (FlnA), actinin 1 (Actn1) and spectrin, that crosslink F-actin using a related F-actin binding site, are critical components of the mature platelet cytoskeleton and regulate proplatelet formation and motility.
Platelet specific RhoA deficiency causes macrothrombocytopenia with platelet counts reduced by ~50% from normal. The RhoA-/- platelets have many functional defects that cause animals to readily bleed, but they have normal lifetimes in blood . One pathway by which RhoA deficiency may affect MK maturation and platelet formation is through ROCK modulation of myosin 2a activity in MKs. RhoA activates ROCK, inhibiting myosin LC phosphatase and hence leading to increased myosin LC phosphorylation. This activity is thought to restrain proplatelet elaboration and thus RhoA deficiency to release this brake, causing premature proplatelet formation and leading to enlarged and dysfunctional platelets.
|GP1ba KO||Large, fragile|||
|Filamin A KO||Accelerated proplatelet elaboration, altered DMS||Accelerated||10-20% of normal: Large, fragile|||
|Actinin 1 ABD mutants||Decreased proplatelet number, increased proplatelet thickness||Reduced, thickened||Large|||
HC IIA KO
|Increased, formation accelerated||Heterogenous,|
Reduced by 70%
|Dynamin 2 KO||Reduced, thickened||20% of normal, size highly variable|
|Pacsin 2 KO||Normal|
|β1-tubulin||Compensate by upregulating β2 and β5-tubulin||Proplatelet formation blocked||Amorphic, reduced by 70-75%|||
|ADF||normal||90% of normal,|
|ADF/Cofilin1||Numbers increased 3X; DMS and cytoplasmic structure altered||decreased||5% of normal count; sizes variable, amorphic|||
|Actin interacting protein|
|Defective megakaryo- poiesis: small, DMS abnormalities||20% of normal: large||Normal|||
|Profilin 1||Large, size variable|||
|RhoA||50% of normal, large -125%||Slight increase in turnover rate|||
|Spectrin||Proplatelets disrupted by dimer-dimer self-association inhibitor|||
|Tmod3||Altered DMS||Enlarged tips||Large, decreased count|||
Cofilin is regulated by Aip1, a protein that binds and enhances filament severing /actin depolymerization activity. The importance of this protein to platelet production has been demonstrated . N-ethyl-N-nitrosourea mutagenesis generated mice with profound thrombocytopenia, bleeding, and enlarged, amorphic platelets contain a gene mutation that was mapped to Wdr1 gene. The high degree of similarity to the phenotype of in the ADF/cofilin double knockout, strongly suggests both proteins are in the same thrombogenesis effector pathway.
In certain ways, the profilin 1 KO phenotype is similar to the behavior of platelets in Wiskott-Aldrich syndrome, or in WASp or WIP KO mice. WASp is a signaling protein promotes actin assembly system, binding to Arp2/3 when activated by binding of GTP-cdc42. However, the bulk of studies performed on human WAS platelets or platelets derived from KO mice revealed normal and ever hyper-function for the WASp null platelets. Human patients have low platelet counts that are caused by accelerated platelet clearance, a process that lowers the platelet count in the Wasp/Wip KO mice. Recently studies have shown the MT ring in platelets from WAS patients to be hyper-acetylated, like the profilin 1 KO platelets. However, in the WAS syndrome, platelets in the circulation are small, not large, as is found in the Profilin null animals.